JP2005530151A - Method and apparatus for low resistance electrophoresis of precast hydratable separation media - Google Patents

Abstract

Methods and apparatus are provided that facilitate electrophoresis of precast hydratable separation media, typically immobilized pH gradient (IPG) strips. The method utilizes the swelling of the precast hydratable separation medium upon rehydration to accommodate the medium in an encapsulating member (10) that allows for electrical contact with the encapsulated separation medium. help. Electrical contact allows a voltage gradient sufficient to perform analyte separation to be established in the enclosed separation medium. A cassette (100), buffer core (126), electrophoresis system and kit for implementing the method of the present invention are provided.

Description

(Description of related applications)
This application claims priority from US Provisional Application No. 60 / 390,259, filed Jun. 18, 2002, and is prior to US Provisional Application No. 60 / 290,464, filed May 10, 2001. This is a continuation-in-part of US patent application Ser. No. 10 / 102,188 filed Mar. 18, 2002 claiming priority, the disclosures of which are incorporated by reference in their entirety.

(Field of Invention)
The present invention relates to a method and apparatus for electrophoresis of precast hydratable separation media. Specifically, the present invention relates to methods, cassettes, buffer cores and systems useful for performing isoelectric focusing using immobilized pH gradient (IPG) strips.

  Isoelectric focusing (IEF) has been used for over 30 years as an important means of analyzing proteins in complex mixtures such as proteins present in biological samples.

  In isoelectric focusing, proteins are generally driven by an electric field applied to a pH gradient established in a carrier matrix such as a gel. The protein moves until its isoelectric point (pI) matches the local pH. At the point of coincidence, the protein no longer has a net charge and stops moving. Proteins are concentrated at points characteristic of that protein.

  In the original method, a pH gradient for IEF was established and maintained in the gel matrix by mobile carrier ampholyte (CA). In general, when a gel is polymerized and a voltage gradient is applied in the presence of a population of CAs having a range of charge characteristics, a pH gradient is established in the gel by aligning various CA species in the matrix.

  IEF with CA has proven to be very useful, but soon the pH gradient created by CA is susceptible to titration with atmospheric carbon dioxide, and over time, the migration of CA to the cathode and the pH A phenomenon called cathode drift that led to the breakdown of the gradient was discovered.

Cathode drift can be reduced by casting into a tube encapsulating IEF gel and thus limiting contact with atmospheric CO ₂ . However, the tube traps prepolymer component impurities in the matrix during polymerization and prevents separation. Furthermore, the tube format causes difficulties when second dimension separation such as size fractionation is desired.

  Bjellvist and colleagues addressed the problem of cathode drift in a different manner and now fixed the pH gradient in the support matrix by a technique called immobilized pH gradient (IPG) isoelectric focusing. Bierkvist et al., J. Biochem. Biophys. Methods, Vol. 6, pp. 317-39 (1982), Rigetti et al., Trends in Bio. Chemical Sciences (Trends Biochem. Sci.), Vol. 13, No. 9, 335-8 (1988), Righetti et al., Methods in Enzymology, Vol. 270, 235-55 (1996), US Pat. No. 4,130,470, and Righetti, “Immobilized pH Gradient: Theory and Methodology” (Biochemistry and Molecular Biology) Laboratory techniques in (Laboratory Techniques in Biochemistry and Molecular Biology), Vol. 20), Elsevier life medical publishers (Elsevier Biomedical Press, LTD), Refer to the Netherlands (ASIN 0444813012). Soon, two-dimensional electrophoresis using IPG IEF and size fractionation was developed. Gorg et al., Electrophoresis, Vol. 9, No. 9, pages 531-46 (1988).

  The plastic support plate of the IPG strip imparts sufficient structural elasticity to the gel so that it can be washed before use. This proved that IPG not only reduced the problem of cathode drift, but was also useful in reducing interference from prepolymer component impurities. The increased elasticity also allows the gel to be stored in a dehydrated form before use. Today, many vendors sell IPG dehydration strips with different pH ranges and different separation lengths (eg, Amersham Biosciences, Piscataway, NJ, USA, Piscataway, NJ, USA). ) Immobiline Dry Strip Gels, Ladies Trip IPG (Ready Strip IPG) of Bio-Rad Laboratories, Hercules, Calif., USA (Bio-Rad Laboratories, Hercules, CA, USA).

  But there are still challenges.

Cathodic drift can be prevented by immobilizing the gradient-forming ampholyte, but the charged immobilization moiety (immobilin) is still susceptible to titration by atmospheric CO ₂ . CO ₂ titration is exacerbated by the fact that the separation media IPG strip is in direct contact with air on at least one side. By direct contact with air, the matrix may dehydrate during electrophoresis and the salt may crystallize.

  The response to these problems was partly a methodological solution rather than a structural solution. That is, a solution: plastic-supported IPG strip is generally occluded by an oil layer to exclude air and slow evaporation to perform electrophoresis.

  However, the method of plugging with a layer of liquid oil has its own difficulties. Important among these problems is the need to perform electrophoresis while keeping the IPG strip horizontal. Commonly used for SDS-polyacrylamide gel electrophoresis (SDS-PAGE) such as that described in US Pat. No. 5,888,369 to Tippins et al. Because horizontal orientation is unavoidable. It cannot be used for a vertical electrophoresis apparatus having a small installation area. Furthermore, the use of oil requires detailed procedures and takes time.

  US Pat. No. 6,013,165 to Wiktorowicz et al. Describes an apparatus that can perform immobilized pH gradient isoelectric focusing without the use of a liquid oil layer. A continuous pKa gradient is immobilized on at least one of the major opposing faces of the cavity formed between the two plates. The cavity that can be further partitioned into parallel channels is then filled with a flowable separation medium. Preferably, electrophoresis is performed with the assembly in a horizontal orientation to minimize convection currents in the flowable separation medium. When this apparatus is used, a precast hydratable separation medium such as a commercially available IPG strip cannot be easily inserted, and electrophoresis in the vertical dimension cannot be easily performed.

  Accordingly, there is a real need in the art for methods and apparatus that allow electrophoresis of IPG strips and other precast hydratable separation media without the need for contact with an occlusive liquid oil layer. In addition, there is a need in the art for methods and apparatus that allow IPG strips and other precast hydratable separation media to be electrophoresed vertically.

(Summary of the Invention)
The present invention fulfills these and other needs in the art by providing methods, apparatus and kits for electrophoresis of precast hydratable separation media, thereby eliminating the need for the use of an occlusive oil layer. Avoid the need to run the run horizontally.

  The present invention, in part, utilizes the swelling associated with rehydration of a precast hydratable separation medium to enclose such medium in an enclosure that allows for electrical contact with the enclosed separation medium. Based on the discovery that it can help accommodate. The spaced electrical contact allows a voltage gradient to be applied to the medium while the precast hydratable separation medium is encapsulated except for electricity, and electrophoresis can be performed in the cassette.

  Encapsulated and separation medium contact with air is substantially reduced. When the precast hydratable separation medium is an IPG strip, the need for the prior art for occlusive contact with the fluid oil layer during immobilized pH gradient isoelectric focusing is avoided by reducing air contact.

  Encapsulated and without an associated fluid oil layer, the precast separation media can be electrophoresed in any physical direction. When the precast hydratable separation medium is an IPG strip, the need for prior art for horizontal electrophoresis is alleviated and the vertical footprint currently used to perform widespread SDS-PAGE is small. It is newly possible to perform IPG electrophoresis using an electrophoresis gel box.

  The present invention is further based on a novel device design that minimizes the resistance between the power supply and the gel, so that the parasitic system impedance is reduced so that a lower voltage is used for a shorter period of time, especially in IPG strips. It is possible to carry out isoelectric focusing.

  Accordingly, the present invention provides, in a first aspect: hydrating and housing a precast hydratable electrophoretic separation medium in an encapsulating member that allows for electrical contact with the encapsulated medium; and Providing a method of performing an electrophoresis method comprising establishing a voltage gradient in an encapsulated separation medium sufficient to perform electrophoretic separation of an internal analyte using electrically spaced contacts .

  In one embodiment, the method further comprises a previous step of inserting the precast hydratable electrophoretic separation medium in its dehydrated state into the encapsulating member. In another embodiment, the method further comprises a subsequent step of removing the precast hydratable electrophoretic separation medium from the encapsulating member. For example, a sample that has been one-dimensionally fractionated using the medium once taken out may be applied to the gel to perform second-dimensional separation.

  In certain embodiments, the hydrating and storing step comprises contacting the dehydrated precast hydratable electrophoretic separation medium with an aqueous solution, often an aqueous solution containing a sample to be fractionated.

  The method of the invention is particularly useful for performing isoelectric focusing using immobilized pH gradient strips. Accordingly, the precast hydratable electrophoretic separation media used in the practice of the present invention may typically have an immobilized pH gradient.

  As explained above, the method of the present invention comprises (i) means for hydrating and storing a precast electrophoretic separation medium in an encapsulating member, and (ii) electrical contact having a spacing with the encapsulated separation medium. A voltage gradient is applied to the encapsulated medium sufficient to perform electrophoretic separation of the analyte present in the member using spaced electrical contact means. Add.

  Accordingly, in another aspect, the present invention performs electrophoresis comprising means for hydrating and containing a precast electrophoretic separation medium in an encapsulating member, and means for electrical contact with a spacing from the encapsulated medium. A cassette capable of establishing a voltage gradient sufficient to perform electrophoretic separation of an internal analyte in a separation medium using spaced electrical contact means.

  In some embodiments, the cassette of the present invention includes a shape retention member and at least one channel, which provides dimensional integrity to the channel (s). In a typical embodiment, the cassette includes a plurality of such channels.

  Each channel present in the cassette and useful for performing the method of the present invention has a first channel inlet, a second channel inlet and a cavity between the two inlets, the channel cavity being hydrated The precast electrophoretic separation medium is sized to allow insertion in a dehydrated state and enclose and accommodate the strip in a rehydrated state. The first and second channel inlets allow spaced electrical contact with the channel cavity and allow electrical current to flow through the channel cavity by the spaced electrical contact.

  In one embodiment, shape retaining members are used on all outer walls of the channel cavity. In other multi-layer laminate embodiments, the cassette further includes a laminate cover that attaches directly or indirectly to the shape retaining member to form at least a portion of the channel outer wall. In these latter embodiments, the adhesion of the laminate cover to the shape retaining member is generally reversible.

  In other embodiments, the cassette further includes a first well forming member that is directly or indirectly attached to the shape retaining member and includes a fluid reservoir at the plurality of first channel inlets. Define the shape. Typically, the cassette may further include a second well-forming member that is directly or indirectly attached to the shape-retaining member and contains a fluid reservoir at a plurality of second channel inlets. Define the shape. If present, these well-forming members may normally be reversibly adherent to the shape-retaining member.

  In one related series of embodiments, the first and second channel inlets of each channel allow electrical contact with the channel cavity between the two inlets through the common surface of the cassette. In another related series of embodiments, the first and second channel inlets allow electrical contact with the cavity between the two inlets through another surface of the cassette. These two incompatible geometrical arrangements require different electrode geometrical arrangements and thus different electrophoretic buffer cores to complete the circuitry required for electrophoresis.

  The precast hydratable electrophoretic separation media may be provided by the user and may be included in one or more channels of the cassette without requiring user insertion or packaged separately in a kit with the cassette. May be provided.

  With respect to the latter, it is another aspect of the present invention to provide a kit that facilitates electrophoresis of a precast hydratable electrophoretic separation medium. In general, the kit includes a cassette of the present invention and at least one precast hydratable electrophoretic separation medium appropriately sized to be hydrated and contained in said cassette.

  In certain embodiments, the kit includes at least one conductive wick for use with the cassette and the cassette. In many cases, a sufficient number of wicks are provided in such kits to facilitate anode and cathode contact with the cassette.

  The cassettes of the present invention may be used to perform vertical electrophoresis of precast hydratable separation media in a buffer reservoir commonly used for SDS-PAGE electrophoresis, usually with a buffer core. In cassette embodiments where the first and second channel inlets open on separate sides of the cassette, the buffer core currently used for SDS-PAGE may be used. In cassette embodiments where the first and second channel inlets open on the same side of the cassette, an alternative buffer core geometry is required.

  Accordingly, it is another aspect of the present invention to provide a buffer core comprising a substantially rigid frame, an anode, and a cathode in spaced relation with the anode for vertical electrophoresis of precast hydratable electrophoretic separation media. It is the aspect of. The buffer core frame has a first cassette engaging surface and a second cassette engaging surface. Engagement of the first and second cassettes with the first and second frame engagement surfaces, respectively, creates a frame interior chamber sealed with five surfaces. The cathode and anode are in electrical contact with the interior of the interior chamber, respectively, and engage the frame engagement surface by engaging the first and second cassettes with the first and second frame engagement surfaces, respectively. Contact with the spacing of the anode and cathode to the surface of at least one cassette occurs, allowing electrophoresis of the precast hydratable separation medium encapsulated therein.

  The cassette and buffer core system of the present invention reduces the resistance between the power source and the gel, allowing electrophoretic separation using lower voltage, shorter time, lower total volt-time.

  Accordingly, in another aspect, the present invention provides a system for low resistance electrophoresis of analyte samples in a precast hydratable separation media strip. The system includes means for encapsulating a plurality of strips and means for simultaneously achieving a single anode and single cathode spaced electrical contact with each of the encapsulated strips in response to an external compressive force. Including.

  The enclosing means individually allow electrical contact with a distance from each of the encapsulated strips through the first and second inlets, respectively. The electrical contact means can distribute an external compressive force to force the anode and cathode against the encapsulation means at a pressure greater than other locations on the encapsulation means at the first and second inlets.

  The electrical contact means may include an anode support, a cathode support anode, and a cathode. The support in these embodiments discontinuously distributes the external compressive force to the anode and cathode to encapsulate the anode and cathode at the first and second inlets at a greater pressure than elsewhere on the encapsulation means. Press to. In some embodiments, the anode support contacts the anode discontinuously and the cathode support contacts the cathode discontinuously.

  In a general embodiment of the system of this aspect of the invention, the encapsulating means can hydrate and house the strip.

  The system provides a substantially lower resistance path between the power source and the gel for electrophoresis of precast hydratable separation media such as IPG strips than prior art devices.

  The system of this aspect of the invention may be able to perform electrophoretic separations, including isoelectric focusing in IPG strips, even by application of up to 3000 volts or less, 1500 volts or less, 500 volts or less, for example. The system may be able to perform electrophoretic separations including isoelectric focusing in IPG strips even with an application of less than 2000 volts-hour and as much as 1500 volts-hour. The system of this aspect of the invention may be able to achieve electrophoretic separations including isoelectric focusing in IPG strips even in less than 6 hours, less than 5 hours, even less than 4 hours.

  In another aspect, the present invention provides a method for low resistance electrophoresis of an analyte sample in a precast hydratable separation media strip.

  The method hydrates and houses at least one strip in an encapsulating member that allows for electrical contact with individual spacing with each of a plurality of encapsulated strips through first and second inlets, respectively. A step of applying a sample containing a protein analyte to the encapsulated strip, and a step of simultaneously forcing an electrical contact having a distance from each of the encapsulated strips by pressing the anode and cathode against the encapsulating member. Distributing the force pressing the anode and cathode against the encapsulating member to produce a greater contact pressure at the first and second inlets than elsewhere on the encapsulating member, and electrophoresis of the analyte in the encapsulated strip Applying a potential to the anode and cathode with a potential difference for a time sufficient to effect separation.

  In one embodiment, the sample is applied during the containment of the strip in the encapsulating member.

  The method provides a resistance path between the power source and the isolation medium that is substantially lower than found in the prior art.

  Thus, the method of this aspect may obtain effective separation including isoelectric focusing in IPG strips even by application of up to 3000 volts or less, 1500 volts or less, 500 volts or less. The method may perform electrophoretic separations including isoelectric focusing in IPG strips even with the application of less than 2000 volts-hour, only 1500 volts-hour. The method may be able to perform electrophoretic separations, including isoelectric focusing in IPG strips, in less than 6 hours, less than 5 hours, and even less than 4 hours.

  The method of this aspect of the invention may apply the potential difference at multiple ramp voltage steps, multiple stepped voltage steps, or at a constant voltage level.

  Usually the strip is an IPG strip.

  In a related aspect, the present invention is an improved method of electrophoresis using a precast hydratable separation media strip, which allows for electrophoretic separation even at a maximum applied voltage of 3000 volts, 1500 volts, 500 volts or less. A method is provided that includes contacting the strip with the anode and the cathode in a sufficiently low resistance between the power source and the gel.

  In various embodiments of the method of this aspect of the invention, the strip is an immobilized pH gradient (IPG) strip and the resistance is isoelectric by application of nominal power less than 2000 volts-hour, even nominally 1500 volts-hour. Low enough to allow point electrophoresis.

  In yet another aspect, the present invention encapsulates the anode and the cathode in a means that allows separate electrical contact with each of the strips encapsulated through the first and second inlets, respectively. Provided is a buffer core device that forces simultaneous electrical contact with a plurality of precast hydratable separation media strips.

  The apparatus includes a substantially rigid frame, an anode support, a cathode support, an anode, and a cathode. The anode support and cathode support are spaced apart and fixed to the frame to distribute external compressive forces to the cathode and anode, respectively, with greater contact pressure at the first and second inlets than at other locations on the containment means. The cathode and anode can be pressed against the encapsulated strip.

  The anode support can be in intermittent contact with the anode and the cathode support can be in intermittent contact with the cathode.

(Detailed explanation)
The present invention, in part, utilizes the swelling associated with rehydration of a precast hydratable separation medium to enclose such medium in an enclosure that allows for electrical contact with the enclosed separation medium. Based on the discovery that it can help accommodate. The spaced electrical contact allows a voltage gradient to be applied to the medium while the precast hydratable separation medium remains contained within the encapsulating member.

  Encapsulated and separation medium contact with air is substantially reduced. When the precast hydratable separation medium is an IPG strip, the need for the prior art for occlusive contact with the fluid oil layer during immobilized pH gradient isoelectric focusing is avoided by reducing air contact.

  Encapsulated and without an associated fluid oil layer, the precast separation media can be electrophoresed in any physical direction. When the precast hydratable separation medium is an IPG strip, the need for prior art for horizontal electrophoresis is alleviated and the vertical footprint currently used to perform widespread SDS-PAGE is small. It is newly possible to perform IPG electrophoresis using an electrophoresis gel box.

  Accordingly, the present invention provides, in a first aspect, a method for performing electrophoresis, and in particular, a method for performing electrophoresis using a precast hydratable separation medium. As used herein, the term “electrophoresis” explicitly includes isoelectric focusing.

  In the first step, the method includes hydrating and housing the precast hydratable electrophoretic separation medium in an encapsulating member that allows for electrical contact with the encapsulated medium. In the second step, a sufficient voltage gradient is applied to the encapsulated medium to achieve electrophoretic separation of the internal analyte using spaced electrical contacts.

  As used herein, the phrase “precast electrophoretic separation medium” (also “precast separation medium”) refers to an electrophoretic separation medium, generally first solidified or gelled in a different location from within the encapsulating member performing the electrophoresis. Polymer gel.

Electrophoretic separation media and methods for preparing electrophoretic media, methods for casting electrophoretic media, and methods for performing electrophoresis using electrophoretic media are well known in the analytical arts and are described herein. There is no need to elaborate. For example, in the present specification, the entire disclosure of which is incorporated by reference, Rabiraudo (Rabilloud) (ed.), "Proteome Research two-dimensional gel electrophoresis and identification methods (Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods) ", Springer publishing (Springer Verlag), 2000 years (ISBN 3540657924), Wesutamaia (Westermeier) al.," practical electrophoresis (electrophoresis in practice) ", second edition, John Wiley & Sons, Inc. (John Wiley & Sons), 2000 (ISBN 3527300708), BD Heimuzu (B.D.Hames) et al. (Eds.), "Protein gel electrophoresis (Gel Electrophoresis of Proteins)", Third Edition, Oxford University Press (Oxford University Press), 1998 years) (ISBN 0199636419), and Jones (Jones), "gel electrophoresis nucleic acid essential technique (gel electrophoresis: nucleic Acids: essential techniques, ( John Wiley & Sons, Inc., 1996) refer to the (ISBN 0471960438).

  Although polyacrylamide (ie, the polymerization product of acrylamide monomers cross-linked with N, N'-methylenebisacrylamide) and agarose are the two most commonly used polymer gels currently in electrophoresis, the present invention is much more Have proved useful for electrophoresis of various polymer gels.

  Since the gel first solidifies or gels in a location separate from the encapsulating member that performs electrophoresis, the “precast electrophoresis separation medium” used in the present invention is transported or removed from the casting mold and then It must have sufficient structural elasticity to be accommodated in the encapsulating member of the present invention.

  Generally, such structural elasticity is imparted to the separation media by adhesion to or incorporation into a layer or sheet of another material such as plastic. Such layers are known in the art and include, for example, polyester film fabrics that can be incorporated into polyester film supports and separation media, as found in commercially available IPG strips.

  In general, the “precast electrophoretic separation medium” used in the present invention is in the shape of a strip—that is, the first dimension is substantially larger than the second dimension—but such dimensions are useful in the practice of the present invention. Not required. Nevertheless, for ease of explanation, all precast electrophoretic separation media useful in the practice of the present invention are referred to herein as “strips”.

  “Precast hydratable electrophoresis separation media” refers to precast electrophoresis that can be dehydrated and retains sufficient structural integrity to allow electrophoretic separation of internal analytes after rehydration. It is a medium.

  Neither complete removal of water during dehydration nor complete liquid saturation during rehydration is required or intended. If the precast hydratable electrophoretic separation medium is contacted with an aqueous solution (“buffer aqueous solution”, “buffer solution”) in a dehydrated state, it swells to a detectable level, which is sufficient for practicing the present invention.

  Generally, the precast hydratable electrophoretic separation medium is at least about 5% by volume upon contact with a buffered aqueous solution, often at least about 10%, 15%, 20%, at least about 25%, 30% by volume. , May even expand over 40%. The increase in volume may extend to all three dimensions, or may be primarily one or two dimensional when the separation medium is supported by an inextensible layer. The increase in volume may occur in minutes, or more generally in hours in the case of IPG strips.

  When the precast hydratable electrophoretic separation medium comes into contact with an aqueous solution (“buffer aqueous solution”, “buffer solution”), it swells if expanded sufficiently to allow hydratable accommodation in the encapsulating member The degree is sufficient.

  “Hydratable containment” means that the precast hydratable separation medium can be inserted into the encapsulating member in a dehydrated state and can be contained in the enclosing member in a rehydrated state. Intended.

  Although the strip must be “insertable” in the dehydrated state, the strip need not necessarily be removable from the encapsulating member in the dehydrated state.

  When two conditions are met, the rehydrated precast hydratable separation medium is said to be “contained” in the encapsulating member (also the same as “contained and encapsulated” inside). First, the strip remains in the encapsulating member when the enclosing member is oriented vertically. Second, at least 50% of the separation medium is not in direct contact with the surrounding atmosphere when the encapsulating member is oriented vertically. Furthermore, the frictional force and surface tension between the rehydrated separation medium and the encapsulating member may contribute to the internal containment of the strip, but so to implement the containment of the strip in the encapsulating member. It is not intended that a simple frictional force or surface tension is sufficient.

  The encapsulating member is sufficiently shape-retaining to maintain dimensional integrity when in contact with the expanding precast hydratable separation medium. In the embodiment described in detail below, the encapsulating member is a cassette having a shape retaining channel cavity for engaging a precast hydratable separation medium therein.

  The encapsulating member further allows for electrical contact with a distance from the encapsulated precast hydratable separation medium. As discussed further below, contact may be direct, such as by passage through the anode and cathode electrodes, or indirect, such as by passage of current through an intermediate polymer layer or wick.

  After containing the precast hydratable electrophoretic separation medium in the encapsulating member, a voltage gradient sufficient to achieve electrophoretic separation of the internal analytical sample using spaced electrical contacts is applied.

  Although described herein as applying a voltage gradient specifically to the separation medium, it will be understood that current can flow through the separation medium and that the method can be similarly described as flowing current through the separation medium. Is done.

  The electrical parameters used are the composition and dimensions of the encapsulated electrophoretic medium, the composition of the sample, the composition of the rehydration aqueous solution, the desired separation type, and the electrical contact with the spacing with the encapsulated separation medium. Depending on the method to be performed, it can easily be determined empirically by routine experimentation. Specific electrical parameters for isoelectric focusing using the cassette-immobilized IPG strip and buffer core adapter of the present invention are further described herein below.

  Returning to more detailed methods, generally the precast hydratable electrophoretic separation medium is inserted into the encapsulating member in a dehydrated state by the user.

  For example, in an embodiment described further below, a precast hydratable separation medium such as an IPG strip is manually inserted into a channel cavity present in the encapsulating member. As another example, if the encapsulating member comprises a hinge or is otherwise reversibly separable, a precast hydratable separation medium such as an IPG strip is manually removably inserted into the well and the member is then removed. Closing completes the channel cavity.

  Removable insertion of the dehydrated strip into the encapsulating member is common but not always necessary. For example, a dehydrated strip may be inserted earlier during manufacture of the encapsulating member, eliminating the need for the user to insert dehydrated precast separation media into the encapsulating member.

  The dehydrated separation medium is then contacted with an aqueous solution.

  The composition of the aqueous rehydration solution depends on the composition of the sample and the separation medium and the intended electrophoresis procedure, and therefore its choice depends on factors well known in the electrophoresis art.

  For example, if the precast hydratable separation medium is a commercially available IPG strip such as Immobilin Dry Strip (Amersham Biosciences, Inc., Piscataway, NJ), the rehydrating aqueous solution is usually urea, non- Carrier ampholyte mixtures suitable for the pH range of ionic or zwitterionic surfactants, dithiothreitol (DTT), dyes and IPG strips may be included. Carrier ampholyte mixtures for such rehydrating aqueous solutions are commercially available (for example, IPG buffer pH 3.5-5.0 from Amersham Biosciences, Piscataway, NJ, USA, catalog number 17-6002-02, IPG buffer pH 4.5-5.5 with catalog number 17-6002-04, IPG buffer pH 5.0-6.0 with catalog number 17-6002-05, IPG buffer pH 5 with catalog number 17-6002-06 5-6.7, IPG buffer pH 4-7 with catalog number 17-6002-86, IPG buffer pH 6-11 with catalog number 17-6002-78, IPG buffer pH 3- with catalog number 17-6002-88 10NL, catalog number 17-6002-87 IPG buffer pH 3-10).

  The aqueous rehydration solution may also advantageously contain a sample intended to be separated in a precast hydratable separation medium.

  For example, when the precast hydratable electrophoretic separation medium is an IPG strip, the sample to be separated may be a mixture of proteins such as those derived from biological samples, usually denatured by chaotropes, reducing agents and surfactants. Alternatively, it may be already modified. If the separation medium is other than an immobilized pH gradient strip, the sample may contain other types of macromolecules such as nucleic acids.

  The method of the present invention may include a post-step of removing the precast hydratable separation medium from the encapsulating member after electrophoresis. As described further below, the method of removal depends on the structure of the encapsulating member. In some embodiments, instead of removing the separation medium of the method of the present invention, further analysis may be performed in the encapsulating member, for example by staining and drying.

  As explained above, the method of the present invention is for (i) means for hydrating and containing the precast electrophoretic separation medium therein, and (ii) for electrical contact with a spacing with the enclosed separation medium. Encapsulation having means capable of applying a sufficient voltage gradient to the encapsulated separation medium to perform electrophoretic separation of the analyte present in the separation medium using spaced electrical contact means Including the use of components.

  Accordingly, it is another aspect of the present invention to provide an encapsulating member useful in the practice of the method of the present invention, which is hereinafter referred to as a “cassette”.

  1A-1C are front perspective views of an embodiment of the cassette of the present invention.

  The cassette 100 includes a shape retaining member 10 and at least one channel 12 (in the embodiment shown in FIGS. 1A and 1B, the cassette 100 has six substantially parallel channels 12, although fewer or less More than this). The shape retaining member 10 imparts dimensional integrity to the pre-formed channel 12. In FIG. 1C, the channel 12 is present but not visible because it is completely opaque.

  Referring again to FIG. 1A, the channel 12 has a first channel inlet 14 and a second channel inlet 16 and a cavity 18 between these two inlets. The cavity 18 of the channel 12 is removably engaged with a precast hydratable electrophoretic medium (“strip”), such as an IPG strip, in a dehydrated state, and is dimensioned to contain and enclose the strip after hydration of the strip. Is set.

  The first channel inlet 14 and the second channel inlet 16 allow electrical contact with the cavity 18 and thus define the channel current flow axis through the cavity 18. In some embodiments of cassette 100 (see below) specifically designed for use with prior art buffer cores, the channel current flow axis is substantially in a substantially planar first surface of shape retaining member 10. In parallel planes.

  In order to use the cassette 100 in the method of the present invention, a rehydratable electrophoresis strip 20 such as an IPG strip is generally inserted through the inlet 14 or inlet 16 into the channel 12 in a dehydrated state. In an alternative embodiment, the strip 20 is pre-inserted into the cassette 100 by the user or by the strip manufacturer.

  The strip 20 is rehydrated in the channel 12 by the application of an aqueous rehydration solution that optionally contains the sample to be fractionated.

  Since wetting of the inside of the channel 12 facilitates the insertion of the strip 20 into the channel 12, the aqueous rehydration solution is generally injected into the channel 12 prior to insertion of the strip 20. However, the strip 20 may be pre-inserted into the channel 12 and then rehydrated aqueous solution injected at one or both of the inlets 14 and 16. For samples that require a long rehydration time, inlet 14, inlet 16 or both may be sealed—for example, with tape or a cover glass—to prevent evaporation and accidental release of the aqueous rehydration solution.

  When the strip 20 is rehydrated, it is at least partially contained (secured) in the cavity 18 of the channel 12 by swelling of the separation medium. As will be described further below, the strip 20 is then not easily removed from the channel 12 without expanding the cavity 18.

  If the sample to be electrophoresed and fractionated is not contained in the aqueous rehydration solution, the sample is then leveled to inject the sample into inlet 14, inlet 16 or both in order to hold the sample. Get into the medium. Alternatively, the sample may be preliminarily absorbed into the wick, the wick may be inserted into the inlet 14, the inlet 16, or both, and the sample may enter the separation medium from this wick. As will be described further below, current introduction may be facilitated to facilitate sample introduction.

  Next, a voltage gradient is applied to the strip 20, and current is passed along the flow axis of the channel current to perform electrophoresis.

  Thereafter, the strip 20 is typically removed from the channel 12 for subsequent processing, such as contacting the strip 20 (or a portion thereof) with the gel to perform a staining step and / or a second dimension separation. In general, removal is performed by expanding the cavity 18 using a method suitable for the construction of the cassette 100. For example, in embodiments of the cassette 100 where one or more lamellas constitute the outer wall of the cavity 18, removal may be performed by peeling the lamella and thus opening the channel 12. Depending on the purpose, subsequent processing may be performed in the channel 12.

  Returning to FIG. 1A, the shape-retaining member 10 is constructed of a shape-retaining non-liquid material. Preferred materials are easily machined, molded, or etched, chemically compatible with electrophoresis buffer systems-i.e., do not substantially degrade upon contact, and have little binding to analytes in the encapsulated gel or It does not impede movement and provides a vapor gas barrier. Typically, the shape-retaining member 10 may be constructed from a transparent material including translucent material or optical quality transparent material so that the strip 20 is visible while engaged in the cavity 18. Generally, the shape-retaining member 10 is constructed of a material that is substantially electrically non-conductive, thus reducing or eliminating simultaneous effects on the strip 20 of the electric field other than along the channel current flow axis in the cavity 18. .

  In a typical embodiment, shape retaining member 10 is composed of ceramic, quartz, glass, silicon and derivatives thereof, plastic, or a mixture thereof. Plastics useful for the construction of the shape retaining member 10 include polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethyl methacrylate, polyvinyl chloride, polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal, polysulfone, cellulose acetate, and nitric acid. There are cellulose, nitrocellulose, polystyrene, polyacrylonitrile, polyurethane, polyamide, polyaniline, polyester, and mixtures or copolymers thereof.

  Typically, the shape retaining member 10 is also constructed of a material that allows heat to be removed from the strip 20 by conduction during electrophoresis. For that matter, as will be discussed further below, the shape-retaining member 10 is typically shaped to include one or more recessed areas 27 as shown in FIGS. May reduce the heat transfer resistance between the strip 20 and a heat sink, usually a fluid-filled chamber.

  The shape retaining member 10 imparts dimensional integrity to the channel 12. In order to allow the rehydration aqueous solution (with optional analyte to be fractionated) into the channel 12, to allow the strip 20 to be inserted into the channel 20, and to hydrate in the channel 12. The dimensional integrity is important in order to realize the accommodation and hydration of the strip 20.

  The shape retaining member 10 may provide dimensional integrity to the channel 12 by configuring the outer wall of at least a portion of the cavity 18 of the channel 12.

  For example, the cavity 18 of the channel 12 may be constructed as a tunnel, hole, or conduit in the shape retaining member 10. In such an embodiment, the shape retaining member 10 constitutes the entire outer wall of the cavity 18.

  Alternatively, the cavity 18 may be partially enclosed in the shape retaining member 10 so that only a part of the cavity wall on the outer periphery of the cavity 18 is constituted by the member 10. In these latter embodiments, the channel 12 may be machined into the shape-retaining member 10, or depending on the composition of the shape-retaining member 10, lithographic processing, engraving, isotropic or anisotropic etching, milling The shape holding member 10 may be manufactured by processing, mechanical or chemical polishing, or molding. Alternatively, in these latter embodiments, the channel 12 may be fabricated on the shape retaining member 10 from silicon or resin deposits or slabs.

  In embodiments where the cavity 18 is not completely encapsulated by the rigid member 10, the channel 12 fluidly encapsulates the cavity 18 by physically attaching one or more new sheets to the shape retaining member 10. It may be.

  FIG. 4 is an exploded side perspective view of a multilayer laminate embodiment of the cassette 100 of the present invention.

  In the embodiment shown in FIG. 4, the shape retaining member 10 includes a recess 13. The laminate cover 42 includes a plurality of inlets 50. When the laminate cover 42 is attached to the shape retaining member 10, the recess 13 fluidly encloses the cavity 18, so that the inlet 50 becomes the channel inlets 14 and 16 and completes the channel 12.

  As with the shape retaining member 10, the laminate cover 42 may typically be optically translucent or transparent and is typically substantially electrically insulating.

  Similar to the shape retaining member 10, the laminate cover 42 may be composed of ceramic, quartz, glass, silicon and derivatives thereof, alumina, polymer, plastic, or a mixture thereof. Plastics useful for the construction of laminate covers include polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethyl methacrylate, polyvinyl chloride, polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal, polysulfone, cellulose acetate, cellulose nitrate, There are nitrocellulose, polystyrene, polyacrylonitrile, polyurethane, polyamide, polyaniline, polyester, and mixtures or copolymers thereof.

  The laminate cover 42 may typically be flexible, and in many cases is preferably flexible. The laminate cover 42 can be of any thickness, but the laminate cover 42 may typically be a film to provide flexibility.

  The laminate cover 42 may be attached to the shape retaining member 10 by bonding means known in microfabrication techniques including thermal welding, ultrasonic welding, and the use of adhesives or adhesive layers.

For example, US Pat. Nos. 5,800,690 and 5,699,157, which are incorporated herein by reference in their entirety, are planar with thermal welding, application of an adhesive, or natural adhesion between two components. A method is described for completing a channel by attaching a cover component to a micromachined substrate. U.S. Pat. No. 5,593,838, which is incorporated herein by reference in its entirety, describes the application of localized electric fields to silicon (about 1400 ° C.) or Corning 7059 glass (about 844 ° C.). It teaches that melt attachment of cover parts is possible at about 700 ° C., well below the flow temperature. WO 96/04547 (Lockheed Martin Energy Systems) specification, which is incorporated herein by reference in its entirety, is processed after processing in dilute NH ₄ OH / H ₂ O _2. Teach that the cover plate can be attached directly to the glass substrate by annealing at 500 ° C., well below the fluidization temperature of the silicon-based substrate. WO 98/45693 (Aclara Biosciences), which is incorporated herein by reference in its entirety, describes a thermal coupling method for fabricating a microchannel structure encapsulated in a polymer substrate, particularly a plastic substrate. Discloses an adhesive method in which an adhesive is applied with a film having a thickness of 2 μm or less, and a method in which a curable fluid adhesive is rendered non-flowable by partial curing prior to attachment of a member to be bonded.

  Usually, the laminate cover 42 is attached to the shape holding member 10 by a reversible attachment means. Therefore, the user can remove the laminate cover 42 from the shape holding member 10 after the electrophoresis is completed. The strip 20 can then be removed from the channel 12 for subsequent processing. By constructing the laminate cover 42 as a flexible film, the advantage of removal of the laminate cover 42 by the user from the shape retaining member 10 is provided.

  In the embodiment shown in FIG. 4, the laminate cover 42 is adhered and attached to the shape holding member 10 using the double-sided laminate adhesive layer 46.

  As shown, the double-sided laminated adhesive layer 46 has an elongated slot 48 that coincides with the recess 13. Such a slot 48 does not cause contact between the double-sided adhesive layer 46 and the strip 20 when the strip 20 is removably inserted into the channel 12. Contact with the adhesive may interfere with movable insertion of the strip 20 into the cassette 100.

  In a multi-layer laminate embodiment of the cassette 100 where the laminate cover 42 is attached by a double-sided adhesive layer 46, the thickness of the adhesive layer 46 is adjusted to change the inside diameter of the cavity 18 of the channel 12, thereby varying the hydration of various thicknesses. Possible strip media may be accepted.

  In an alternative embodiment of the multi-layer laminate of cassette 100, the laminate cover 42 itself is generally made in the shape of a shape retaining member that is thicker than the flexible film described above. In some of these embodiments, the laminate cover 42 is formed as a separate structure. In other embodiments, the shape-retaining laminate cover 42 and the shape-retaining member 10 are detachably attached to each other with one or more hinges that exist between them. The hinge need not be in the form of a separate intermediate structure, but instead may be in the form of a foldable seam between the shape retaining member 10 and the laminate cover 42. Such seams are common, for example, in plastic cases designed for storing drill bits.

  When the laminate cover 42 is shape-retaining, the laminate cover 42 may be incorporated into the shape-retaining member 10 by fitting the laminate cover 42 into the shape-retaining member 10, for example. The pressure-adaptive surface on the shape retention member 10 and / or the laminate cover 42 facilitates sealing between the two layers and forms an encapsulation member suitable for electrophoresis. The assembly by fitting the laminate cover 42 to the shape holding member 10 has been particularly described. However, the same effect can be obtained by using any other mechanical engagement method such as misconnection or slot / tab engagement. .

  In the multi-layer laminate embodiment of the cassette 100—both with a flexible laminate cover and with a shape-retaining laminate cover—the inner diameter of the cavity 18 by adjusting the depth of the recess of the channel 12 into the shape-retaining member 10. May be adjusted. In a multi-layer laminate embodiment of cassette 100 where laminate cover 42 is thicker than the membrane, the inner diameter of cavity 18 may be further adjusted by adjusting the depth of the recess of channel 12 into laminate cover 42.

  Channel 12—both in the multilayer laminate and integral embodiment of cassette 100—allows dehydrated insertion of electrophoretic media based on precast hydratable strips such as IPG strips and accommodates the strips after hydration And make the size necessary to enclose.

  The immobilin dry strip IPG strip currently available from Amersham Biosciences (Piscataway, NJ) has a width of about 3 mm and a depth of 0.5 mm. Thus, to enable electrophoresis of these commercially available IPG strips, the channel 12 of the cassette 100 removably engages such strips in a dehydrated state and accommodates the strips when rehydrated. At least about 3.0 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm and 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm for encapsulation And at least about 0.5 mm, 0.6 mm, 0.61 mm, 0.62 mm, 0.63 mm, 0.64 mm, 0.65 mm, 0.66 mm, 0.67 mm, 0.68 mm 0.69mm, and 0.7mm, 0.71mm, 0.72mm, 0.73mm, 0.74mm, 0.75mm, 0.76mm, and 0.77mm It has a depth that.

  The Ladies Trip IPG strip currently commercially available from Bio-Rad (Hercules, CA, USA) has a strip width of 3.3 mm and a gel thickness of 0.5 mm. Thus, to allow electrophoresis of these commercially available IPG strips, the channel 12 of the cassette 100 removably engages such strips in a dehydrated state and accommodates the strips when rehydrated. Approximately at least about 3.3 mm, 3.4 mm, and 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm, and 4.1 mm for encapsulation Width to reach, at least about 0.5 mm, 0.6 mm, 0.61 mm, 0.62 mm, 0.63 mm, 0.64 mm, 0.65 mm, 0.66 mm, 0.67 mm, 0.68 mm, 0.69 mm, and It has a depth as high as 0.7 mm.

  In a presently preferred embodiment suitable for electrophoresis of strips from both manufacturers, channel 12 has a width of 3.7 mm and a depth of 0.64 mm.

  As expected, precast hydratable electrophoretic separation media may be manufactured with dimensions different from those currently used, and are likely to be manufactured. Accordingly, the cassette 100 of the present invention is not limited to a size that is necessary for use with the strip described above.

  The design of the internal dimensions of the channel 12 to allow insertion of media based on a precast hydratable strip in a dehydrated state and to accommodate and enclose the strip when rehydrated is well known in the art. Within the technical scope of

A simple test for qualification of the internal dimensions of channel 12 for a precast hydratable electrophoretic separation medium 20 of any given depth and width is as follows.
(1) Place the cassette horizontally and fill channel 12 with water.
(2) The strip 20 is manually inserted into the channel 12 from the inlet 14 or the inlet 16, and inserted as far as possible.
(3) Eight hours later, the cassette 100 is vertically observed for observation.

  Strip 20 is advanced into channel 12 to the point where less than 1 cm of strip 20 remains outside the inlet selected for insertion in step (2), and more than 50% of the separation medium encapsulated in step (3) If the air is not in direct contact, the channel 12 dimensions are qualified. The strip 20 should also remain contained in the cassette once the cassette is vertical in step (3).

  At one end of the range of usable channel dimensions, the separation medium contacts the inner wall of the channel in direct contact along substantially all of the channel cavity due to swelling of the separation medium. In this case, visible label aqueous solution (eg 0.2 wt / vol% bromphenol blue in water) added to the upper channel inlet is substantially excluded from the channel cavity. That is, the visible label aqueous solution generally penetrates only about 0.25 cm from the channel inlet into the channel cavity. At the other end of the usable channel size range, the swelling of the separation medium is insufficient for the separation medium to make intimate contact with the channel inner wall along substantially all of the channel cavity. In this latter case, when the cassette is vertical, an aqueous visible label solution, such as 0.2% w / v bromophenol blue in water, enters the channel cavity from the top inlet. However, in either case, air does not contact more than 50% of the encapsulated separation medium.

  Another functional test for qualification of the internal dimensions of the channel 12 for a pre-sized hydratable electrophoretic separation medium of a given size replaces the test step (3) shown above with an actual electrophoresis experiment. That is. If strip 20 is advanced into channel 12 to the point where less than 1 cm of strip 20 remains outside the inlet chosen for insertion in step (2), and if appropriate electrophoretic separation is achieved after electrophoresis, channel 12 The dimensions are qualified.

  If the strip 20 is an IPG strip, this latter test can usually be carried out as follows.

  Servo IEF standard solution (Cat. No. 39212-01, Zelba Electrophoresis GmbH, Heidelberg, Germany) of the following composition sufficient to fill the channel: Mix with rehydration buffer. 8.0 M urea, 0.5% ampholyte (Amersham Biosciences catalog number 17-6001-11 3-10 IPG buffer), 2.0% (weight / volume) CHAPS, 20 mM DTT, 0. 0025% (weight / volume) bromophenol blue. Place the cassette horizontally and pipet the aqueous solution into the channel of the cassette. Insert the strip into the channel so that about 3 mm exits the channel entrance. Seal the channel inlet with cover tape and rehydrate the strip for 8-16 hours. Remove cover tape and sample introduction well, if any.

  An electrode wick (described further below) is attached to each inlet set. Apply 750 μL of deionized water uniformly to each electrode wick.

  The cassette is brought into contact with the electrode of the buffer core (described further below). A buffer dam (described further below) is attached to the contact surface on the opposite side of the buffer core. The buffer core is slid into the electrophoresis chamber, and the outer chamber surrounding the buffer core is filled with water. Care is taken not to allow water to spill over the height of the cassette into the inner chamber (the outer wall is defined by the cassette and buffer core).

  Voltage is applied in three steps according to the following profile. 20 minutes at 200V, 15 minutes at 450V, 15 minutes at 750V, 30 minutes at 2000 volts.

  If discrete marker bands can be observed, then the channel dimensions are acceptable.

  Although not necessarily, for example, as shown in FIG. 2, the spacing between the channel inlets 14 and 16 is generally set so that the channel 12 engages substantially the entire length of the strip 20.

  Various lengths of IPG strips are currently commercially available. For example, Immobilin dry strip IPG strips currently available from Amersham Biosciences (Piscataway, NJ) are available in gel lengths of 70 mm, 110 mm, 130 mm, 180 mm and 240 mm. Ladies Trip IPG strips currently available from Bio-Rad, Inc. (Hercules, Calif., USA) are available in gel lengths of 70 mm, 110 and 170 mm. Zoom® (ZOOM®) IPG strips currently available from Invitrogen (Carlsbad, Calif., USA) (Invitrogen, Carlsbad, Calif., USA) have a gel length of 70 mm.

  Thus, in the presently preferred specific embodiment of the cassette 100 of the present invention, the channel 12 is shaped to accommodate the entire length of the strip having a gel length of 70 mm, 110 mm, 170 mm, 180 mm and a length of 240 mm. .

  In such commercially available IPG strips, the polyester substrate generally overhangs the gel slightly at both ends. Thus, in general, channel 12 has at least the same length of gel as described (70, 100, 170, 180, or 240 mm) and is generally 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, or 6 mm at both ends. Has an extension that also reaches. Thus, in the case of an IPG strip with a nominal gel length of 70 mm, the channel 12 is at least about 70 mm long, 72 mm long, 74 mm long, 76 mm long, 78 mm long, 80 mm long, It reaches 82mm. In the presently preferred embodiment in the case of an IPG strip with a described gel length of 70 mm, the channel 12 will be 80 mm long.

  As explained above, separation media based on rehydratable strips are expected to be available in various widths and depths in the future and are expected to be available in exactly the same various lengths. Accordingly, it is an aspect of the present invention to provide a cassette 100 having a channel 12 dimensioned to engage with any selected length of precast hydratable electrophoretic separation media.

  As suggested above, it is not preferred if the strip 20 is too long or shorter than the channel 12.

For example, if strip 20 is substantially longer than inlet 14, inlet 16, or both, too long portion (s) of strip 20 will come into contact with atmospheric CO _2, which is an important advantage of the present invention. To disable. Furthermore, the protruding portion (s) of the strip 20 allows leakage from the ampholyte and / or protein strips. Furthermore, only the part of the separation medium that exists between the spaced electrical contacts is available as a function for separation, reducing the functional part of the gel. Finally, the protruding portion (s) can mechanically interfere with the establishment of electrical contacts that need to be done properly for electrophoresis. When the strip 20 is shorter than the channel 12, it may be difficult to establish effective electrical contact with the encapsulated strip.

  To address these issues in cassettes with non-optimal length channels, if the strip 20 is too long than the channel 12, the overlength may be cut off with scissors or a knife. In general, only the part of the strip 20 without the separation medium is cut off. If the strip 20 is too short than the channel 12, the missing end portion may be in effective electrical contact with the exterior of the channel 12 by filling the missing end portion with a conductive channel filling material.

  Typically, the material used to make the tip of the underlength strip 20 in electrical contact with the inlet 14 or 16 of the cassette 100 can be applied in a liquid or semi-fluid state, in which state There are substances that can be shaped within the channel and then polymerize or gel into a shape-retaining phase.

  Usually, the substance may be a polymer gel such as agarose. When used as such, agarose is melted in the presence of an electrolyte-containing buffer, such as an aqueous rehydration solution, applied as a melted liquid to inlet 14, inlet 16, or both, and then spontaneously with a decrease in temperature. It may be gelled. Polyacrylamide may also be used, but in this latter case, the monomer and crosslinker must be polymerized with the addition of a catalyst, as is known in the art.

  Usually, the cassette 100 includes a plurality of channels 12. When the cassette 100 includes a plurality of channels 12, the current flow axes of the plurality of channels 12 are typically substantially parallel to each other, and the cavities 18 of the plurality of channels 12 are mutually connected except for the channel inlets 14 and 16. There is no fluid contact.

  In such an embodiment, the channels 12 need not have the same cavity 18 dimensions, so a single cassette 100 accommodates strips 20 of various dimensions. However, in general, the cavities 18 of the plurality of channels 12 all have the same internal dimensions.

  Although the cassette 100 has been described above as allowing the user to insert the strip 20 into the channel 12, providing a cassette in which the strip 20 is already inserted at the time of manufacture as described above. This is another aspect. Usually, such a cassette 100 may be disposable.

  In order to facilitate sample application, and particularly to facilitate sample application without cross-contamination between the plurality of channels 12, the cassette 100 may typically include a sample introduction well. FIG. 1A shows one embodiment of such a sample introduction well and FIGS. 1B and 1C show another embodiment of such a sample introduction well.

  Referring to FIG. 1A, a cassette 100 having two well forming members 22 is shown. At each of the six inlets 14 and the six inlets 16, two well forming members define separate reservoirs called sample introduction wells. When the well-forming member 22 is above the shape-retaining member 10 and the cassette 100 is leveled, each sample introduction well has a predetermined maximum amount of fluid that contacts the inlet 14 (or inlet 16) without crossover fluid contact with the adjacent inlet. Can be held.

  When injecting a sample to be fractionated after insertion of the strip 20, the sample introduction well injects a sample of a volume below the maximum reservoir volume separately into the individual wells 14 (and / or 16) without crossover contamination. It becomes possible. If the sample is injected in the rehydration buffer prior to insertion of the strip 20 into the channel 12, the sample introduction well prevents crossover contamination by the sample removed from the channel 12 when the strip is inserted.

  After a sample to be fractionated (such as a protein sample for isoelectric focusing on an IPG strip) enters the separation medium of the strip 20, the inlets 14 are then placed in fluid communication with each other, and then the inlet Placing 16 in fluid communication with each other typically does not cause crossover contamination between channels 12. Therefore, the well forming member 22 may be removable. Such removal may facilitate subsequent use of the conductive wick 24 as shown in FIG. 3B and further described below.

  Since the well forming member 22 is generally removed prior to electrophoresis, the materials from which the well forming member 22 can be constructed are less constrained than the shape retaining member 10 and the laminate cover 42 in the multilayer laminate embodiment of the cassette 100. . In fact, the well-forming member 22 can be from any material that does not substantially chemically react with the aqueous rehydration solution, such as ceramic, quartz, glass, silicon and its derivatives, plastics, natural or synthetic rubber polymers, or mixtures thereof. May be built. Plastics useful for the construction of the well forming member 22 include polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethyl methacrylate, polyvinyl chloride, polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal, polysulfone, cellulose acetate, nitric acid There are cellulose, nitrocellulose, polystyrene, polyacrylonitrile, polyurethane, polyamide, polyaniline, and mixtures thereof. Silicones and their derivatives are also useful.

  In some embodiments, the well-forming member 22 may be composed of a conductive material, which facilitates “active rehydration” of the strip 20. In “active rehydration”, the strip 20 is rehydrated in the presence of a low potential gradient of about 100 V established between the inlets 14 and 16 along the channel current flow axis of the strip 20.

  If active rehydration is desired, the well forming member 22 may be composed of a conductive material such as a carbon impregnated polymer or a carbon doped polymer. After both the strip 20 and the aqueous rehydration solution are placed in the channel 12 (in either order), the cathode is in contact with the first conductive well forming member 22 and the anode is in contact with the second conductive well forming member 22. And a voltage is applied during the rehydration period. The anode and cathode may be electrode bars as found, for example, in MultiPhor (Amersham Biosciences, Piscataway, NJ) or Blue Horizon (Zelva, Heidelberg, Germany) devices. .

  When the cassette 100 is of an integrated type, i.e. having a channel 12 made completely in the shape retaining member 10, the well forming member 22 may be attached to the shape retaining member 10. Alternatively, when the cassette 100 has a multilayer laminate—for example, a channel 12 made with a partially laminated cover 42 —the well forming member 22 may be attached to the laminate cover 42 as shown in FIG.

  FIG. 5 is a side exploded perspective view showing the well forming member 22 attached to the laminate cover 42 using the well forming member double-sided adhesive layer 54. However, with respect to the attachment of the laminate cover 42 to the shape-retaining member 10, the discussion of which is incorporated herein by reference, micromachining including thermal welding, ultrasonic welding and the use of liquid or partially cured adhesives The well forming member 22 may be attached to the laminate cover 42 by various attachment means well known in the art as well as adhesive layer means.

  Alternatively, the well forming member 22 may be attached to the laminate cover 42 by engagement of a facing occlusal surface such as a snap, engagement of a tongue and a groove, or engagement of a tab and a slot.

  Regardless of how they are joined, the well-forming member 22 is typically reversibly attached to the cassette 100 so that the well-forming member can be removed prior to electrophoresis. When the attachment is performed by the well forming member double-sided adhesive layer 54, the adhesive layer is usually adhered more strongly to the shape holding member 10 than the well forming member 22 (or to the laminate cover 42 in the multilayer laminate embodiment). To design. In such embodiments with different bond strengths, generally, when the well-forming member 22 is removed, the adhesive layer 54 remains on the shape-retaining member 10 (or laminate cover 42), and a conductive wick is formed as described further below. Easy to use.

  FIG. 3A shows an embodiment of the cassette of the present invention with the well forming member 22 removed prior to use of the conductive wick, as further described below.

  The introduction of the sample into the separation medium of the strip 20 generally eliminates the possibility of sample cross-contamination, such as between a plurality of channels 12, so that the well-forming member 22 must continue to exist during electrophoresis. Although, it may still be advantageous to further seal the inlets 14 and / or 16 after sample application.

  In these latter embodiments, the inlet 14 and / or the substance that is electrically conductive and can be applied in conformity with the shape of the inlet and / or sample introduction well and then polymerized or gelled into a shape-retaining phase. Sealing is realized by application to 16. As noted above, usefully such materials may be polymeric gels such as agarose or acrylamide.

  In a particularly useful approach, the inlets 14 and / or 16 are sealed with a sufficient amount of material to fill the channel 12 and the inlet 14 (and / or the inlet 16) to the same level as the surface of the shape retaining member 10. Such a geometry facilitates direct or indirect electrical contact between the anode and cathode ends of the strip 20 and the anode and cathode electrodes.

  Returning to FIG. 1A, the cassette 100 may usefully include ribs 40 as needed.

  The rib 40 facilitates the alignment of the laminate cover 42 and the well forming member 22 when the cassette 100 is manufactured. As described further below, the ribs 40 also facilitate proper engagement for operation of the cassette 100 by the electrophoresis chamber or buffer core.

  The ribs 40 may be machined or molded directly from the shape retention member 10 or may be constructed separately and secured to the shape retention material. When constructed separately, it is usefully easily machined, molded, or etched and chemically coexists with the electrophoresis buffer system--that is, hardly decomposes upon contact with the electrophoresis buffer system-solid material or semi-solid Build rib 40 from material. Usefully, the ribs 40 may be constructed of a substantially electrically insulating material including ceramic, quartz, glass, silicon and derivatives thereof, or plastic, or mixtures thereof. Plastics useful for the construction of ribs 40 include polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethyl methacrylate, polyvinyl chloride, polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal, polysulfone, cellulose acetate, cellulose nitrate, There are nitrocellulose, polystyrene, polyacrylonitrile, polyurethane, polyamide, polyaniline, polyester, and mixtures and copolymers thereof.

  As described above, once the strip is rehydrated and the sample is introduced into the strip 20, the strip 20 is received in the cavity 18 of the channel 12. Once the strip 20 has been encapsulated in this manner, a strip of current from the cassette 100 is applied by applying a voltage gradient to the strip 20 along the channel current axis sufficient to achieve electrophoretic separation of the internal analyte. Electrophoresis may be performed without removing 20.

  FIG. 3B illustrates one useful but non-limiting approach for bringing the strip 20 contained in the cassette into contact with the cathode and anode electrodes to complete the necessary electrical circuitry.

  FIG. 3B is a front perspective view of the cassette of the present invention having six channels 12. As shown, the first conductive wick 24 contacts the strip 20 (present in three of the six available channels 12) at the inlet 14 and the second conductive wick 24 is at the inlet 16. To contact the strip 20.

  The wick 24 includes a conductive material. The material need not be structurally conductive. That is, it is sufficient if the wick 24 is conductive when wet, and in fact, typically the wick 24 is conductive when wet. In this latter case, the wick 24 may be usefully composed of a material that readily absorbs moisture, such as paper, nitrocellulose, felt, nylon, or derivatives thereof.

  As described above, the strip 20 may be electrically connected to the cathode electrode and the anode electrode through a conductive polymer or hydrogel such as agarose instead of or in addition to the presence of the wick 24. .

  As shown in FIG. 3B, preferably the first conductive wick 24 may be in contact with each of the plurality of inlets 14, and preferably the second conductive wick 24 is in contact with each of the plurality of inlets 16. In other words, it facilitates the parallel application of current to the plurality of channels 12. While useful, such a geometry is not essential.

  The first conductive wick 24 is then brought into contact with the electrode and used as either the cathode or the anode. The choice of wick 24 as cathode or anode depends on the intended electrophoresis technique, sample application location, and other conditions well known to those skilled in the electrophoresis art. For example, in the case of isoelectric focusing using an IPG strip in which one end of the strip is acidic and the other end is basic, the arrangement is preferably such that the basic end of the strip is electrically connected to the cathode electrode. .

  Next, the second conductive wick 24 is brought into contact with the electrode (the anode if the first wick 24 is in contact with the cathode, and the cathode if the first wick 24 is in contact with the anode).

  Any means of attaching the electrode to the wick 24 may be used as long as an effective electrical connection is ensured.

  As an alternative to using the conductive wick 24, the anode and cathode electrodes may be in direct contact with the strip 20 to make an electrical connection with a spacing from the encapsulated strip 20. Contact may be performed by passing the anode and cathode electrodes through the inlets 14 or 16 or by passing the electrodes through the shape retention member 10 or the laminate cover 42 at a location other than the inlets 14 and / or 16. As an example of the latter approach, in embodiments where the laminate cover 42 is a flexible film, the strips 20 are spaced apart by piercing the laminate cover 42 using electrodes shaped like a razor blade. You may make it contact.

  Thereafter, the electrophoresis may be performed with the cassette 100 placed in an arbitrary physical direction. A particularly useful approach ensures electrode contact with an adapter that allows the cassette 100 to be held vertically and to perform electrophoresis. Even when the cassette 100 is vertical, the channels 12 of the cassette 100 may be horizontal or vertical as desired.

  Returning to FIG. 3B, it is another aspect of the present invention to provide an adapter that allows the cassette 100 of the present invention containing the strip 20 therein to be electrophoresed vertically. It should be noted that the channel 12 may be in the horizontal direction even when the cassette 100 itself is in the vertical direction. In such a direction, if there are a plurality of the channels 12 of the cassette 100, they are located at a certain distance in the vertical direction. Thus, to avoid confusion, the term “vertical” is intended to refer to the direction of the cassette and not the direction of the channel.

  The electrophoresis of the vertical cassette 100 has the obvious feature of reducing the footprint of the electrophoresis apparatus, freeing valuable work space for other devices or applications.

  Furthermore, modular electrophoresis systems that perform slab gel electrophoresis vertically are well known (see, eg, US Pat. Nos. 5,888,369 and 6,001,233) and are commercially available (see FIG. Invitrogen, Carlsbad, CA, USA; Bio-Rad, Hercules, CA, USA). In a preferred embodiment, the adapter of the present invention allows electrophoresis of the cassette 100 of the present invention in such existing modular electrophoresis systems, and electrophoresis of precast hydratable electrophoresis separation media such as IPG strips. Efficient use of such already purchased equipment for use is made possible.

  FIG. 6A is a front perspective view of an adapter referred to herein as a buffer core of the present invention. FIG. 6B is a front perspective view of the buffer core (front) that is not yet in contact with the cassette of the present invention (rear), but is aligned in a ready-to-use position. FIG. 6C shows the state of contact during use. As can be observed, the buffer core 26 is designed by simultaneously aligning the cathode electrode wire 31 with the cathode wick 24 of the cassette 100 and the anode electrode wire 32 with the anode wick 24 of the cassette 100.

  As shown in FIG. 6B, cathode wire 31 is attached to cathode contact 38 at a first end, and similarly anode wire 32 is attached to anode contact 36 at a first end. Contacts 38 and 36 allow for detachable attachment of wires with standard female plugs. As is well known in electrophoretic technology, the other end of such a wire is generally connected to a power source, such as an adjustable power source.

  As also shown, the cathode wire 31 extends from the cathode contact 38 to the support 28 and ends at the second end, and the anode wire 32 extends from the anode contact 36 to the support 30 and ends at the second end. In general, the supports 28 and 30 are made of a material that is substantially electrically insulating and substantially inert to the electrophoresis buffer. For example, the supports 28 and 30 are simply made of plastic such as polycarbonate.

  An external compression force is applied inward to the cassette 100 to realize contact between the anode wire 32 and the cathode wire 31 of the cassette 100 and the respective wicks 24. Thereby, the anode wire and the cathode wire are pressed between the respective supports and the wick to complete an electrical circuit.

  As shown in FIG. 6D, the buffer core 26 may be simultaneously aligned and contacted with the second cassette 100 in a ready-to-use position, and is generally so. When aligned and brought into contact in this manner, the buffer core 26 and cassette 100 open only at the top, and form the shape of an internal chamber 62 that is sealed from outside liquid except at the top. If the number of strips that need to be electrophoresed can be accommodated in a single cassette alone, a “buffer dam” that is similar in size to cassette 100 but without channels 12 is used to complete the internal chamber 62 of the buffer core. May be.

  In order to perform electrophoresis using the cassette and buffer core of the present invention, a plurality of cassettes 100 (or a single cassette 100 and a buffer dam) are aligned and brought into contact with the buffer core 26. This assembly is then engaged in the electrophoresis buffer chamber 34. The buffer chamber compresses a plurality of cassettes 100 (or a single cassette 100 and a buffer dam) by itself or in cooperation with another device to make sealing contact with the buffer core 26. As further described in US Pat. No. 6,001,233, which is hereby incorporated by reference in its entirety, such another compression device may be a cam driven clamp “tension wedge”. Good.

  Alternatively, the buffer core 26 is first loosely engaged within the electrophoresis buffer chamber 34, after which the cassette is aligned, brought into contact with the buffer core 26, and then further compressed.

  In general, the buffer core 26 and a plurality of cassettes 100 (or a single cassette 100 and a buffer dam) are provided by a gasket as shown in FIGS. 6A and 6D, such as a silicone gasket that fits into the groove 70 of the buffer core 26, if necessary. To further promote liquid-tight contact with each other.

  As described above, the buffer chamber 26 and the plurality of cassettes 100 (or a single cassette 100 and a buffer dam) that are hermetically engaged define the interior chamber 62. Unless the fluid level in the electrophoresis chamber 60 overflows the cassette 100, the cathode wire 31 and the anode wire 32 are isolated by the chamber from the fluid present outside the buffer core 26 in the electrophoresis chamber 34 (chamber 60). Is done.

  Thus, the electrophoresis chamber 34 may be filled with any selected solution to a level that does not overflow the cassette 100 and does not affect the electrical circuit. Accordingly, such a fluid may preferably be used as a heat sink to reduce the temperature of the strip 20 as current is passed through the strip 20 within the cassette 100.

  Electrophoresis is performed by connecting the anode and cathode to a power source via contacts 36 and 38. As illustrated in FIG. 11, this may be done conveniently by covering with a cover incorporating the electrode. Preferably, the cover may be designed to be used in the present invention so that it fits into an existing electrophoresis chamber but only interfaces with the buffer core of the present invention.

  A potential difference (voltage gradient) sufficient to perform separation of the analyte in the separation medium of the strip 20 is applied. In embodiments where the strip 20 is an IPG strip, the protein begins to move under the influence of the voltage gradient, and the isoelectric focusing protein moves at a point where the protein's pI matches the pH on the fixed gradient. stop.

  Due to the specific resistance of the various connections between the power supply and the gel, the voltage difference actually achieved by the spaced electrical contacts and the strip 20 is always smaller than that reported by the power supply. In the cassette and buffer core of the present invention, such parasitic impedances are, for example, (i) cable connection to the power supply, (ii) resistance of the cable itself, (iii) contact resistance between the cable and the electrode, (iv) ) The contact resistance between the electrode and the wick, (v) the impedance of the wick itself as a function of the ion concentration in the wet wick, which can be changed during operation by either infiltration or diffusion, (vi) the interface Includes the contact resistance between the wick affected by pressure and the gel surface, (vii) the intrinsic impedance of the gel.

  In another aspect, the present invention provides an apparatus that provides a resistance path between a power source and a precast hydratable separation medium that is substantially lower than that found in prior art devices used for electrophoresis in the prior art. To do. The reduced resistance substantially increases the efficiency with which a precast hydratable separation medium such as an IPG strip can be electrophoresed.

  Of the parasitic impedance, the contact resistance between the electrode and the wick and between the wick and the gel can contribute significantly to the overall voltage drop from the power source to the gel. These contact resistances depend on the respective contact pressure. For any given compression force applied inward to the cassette 100, the magnitude of these electrically effective contact pressures depends on the ratio of forces applied at these locations. Accordingly, the supports 28 and 30 are preferably designed to discontinuously distribute the external compressive force and produce greater contact pressure at the first and second channel inlets than elsewhere on the cassette 100.

  In the first series of exemplary embodiments illustrated in FIGS. 6A-6C, the cassette contact surfaces of support 28 and support 30 are non-planar. That is, the overall shape of a series of protrusions with a plurality of discontinuous recesses in between is defined. The external compression force can be discontinuously distributed to the anode wire 31 and the cathode wire 32 by the jagged surface created in this way.

  In the illustrated embodiment, the indentations are deep enough to create periodic discontinuities in the contact between the electrode wires and the respective supports. Such a depth of depression is not essential.

  The recesses are arranged so that the protrusions in between are aligned with the inlet 14 of the cassette 100 (and the inlet 16 is the same for the other electrode). The indentation is sized to create a protrusion having a size approximately equal to the lateral size of the inlets 14 and 16.

  For example, support to minimize electrically inefficient contact with an embodiment of cassette 100 that can hold six strips 20 (and thus maximize the ratio of electrically efficient contact). The bodies 28 and 30 have six protrusions (on at least one cassette contact surface). Protrusions are positioned in alignment with one of the six inlets 14 (same for inlet 16) of the cassette 100, and the protrusions are preferably sized to be approximately equal to the size of the inlets 14 and 16 in the surface direction.

  In general, the surfaces of support 28 and support 30 that contact the same cassette 100 have the same number of protrusions. However, if two cassettes 100 containing different numbers of strips 20 contact the two cassette contact surfaces, the two cassette contact surfaces on each of the supports 28 and 30 have the same number of protrusions (illustrated in FIGS. 6A-6C). As in the embodiment to do). However, in general, the two cassette contact surfaces have the same number of protrusions.

  In another series of embodiments (not shown), the support 28 and support 30 are not recessed. Instead, the support is non-integral and includes materials with different compressibility. The material with the lowest compressibility is placed at the inlet 14 (same for 16) of the cassette 100 and the material with the higher compressibility is placed at another location on the cassette 100. Different degrees of compression cause a differential distribution of external compression forces, resulting in increased contact pressure at the inlet 14 (and 16).

  Voltage (and current) is applied to the strip 20 by discontinuously applied pressure--for example, by jagged the outward surfaces of the supports 28 and 30 as in FIGS. The efficiency obtained is significantly improved. The voltage (and current) application efficiency to the IPG strip, defined herein as the ratio of the current measured on the same strip 20 with the same sample to a given voltage output from the power source, is With a cassette and buffer core, it can be at least 2 times better, 3 times better, 4 times better, and even at least 5 times better than prior art horizontal oil immersed IPG electrophoresis apparatus. The efficiency can be at least 6 times, 7 times, 8 times, 9 times and even at least 10 times better. The data shown in Examples 2 and 3 herein and plotted in FIGS. 9 and 10 show 2 to 4 times better efficiency than observed using prior art oil immersed flatbed IPG devices.

  Increased electrical transfer efficiency provides significant features.

  Increased efficiency allows IPG IEF to be performed using substantially lower power supply voltages and currents, and allows the use of less expensive power supplies without current limiting means. According to the data shown in Example 3 below, even a simple power supply without adjustment function that can provide only a stepped voltage profile may be used, and there is no need for a power supply that can provide a continuously variable voltage profile. To illustrate.

  Increased efficiency allows the volt-time required for isoelectric focusing to be reached in a shorter run time.

  With the cassettes and buffer cores of the present invention, only 6 hours, 5 hours, 4 hours, only 3.5 hours, 3.0 hours, depending on sample, strip length, strip pH range, and voltage profile. Isoelectric focusing can be performed at 5 hours, 2.0 hours, or as little as 1.5 hours or 1.25 hours. Therefore, isoelectric focusing can generally be performed in 1.25-10 hours, 1.5-9 hours, 1.75-8 hours, 2-7 hours, 2.5-6 hours, and 1-3 hours. .

  Therefore, isoelectric focusing can be performed at least twice as fast as with existing horizontal flatbed oil dipping devices, often at least three times, four times, and sometimes even five times faster. Sometimes isoelectric focusing can be performed at least 6 times, 7 times, 8 times, 9 times, sometimes 10 times faster.

  For example, isoelectric focusing using a continuously variable transformer profile with a continuously variable power supply, a 7 cm IPG gel, a cassette capable of holding six such strips, and a buffer core with a jagged support. Can be implemented.

0 to 175 volts in 15 minutes 175 to 2000 volts in 45 minutes 20 to 30 minutes at 2000 volts.

  Using a stepped power supply, a 7 cm IPG gel, a cassette capable of holding six such strips, and a buffer core with a jagged support, isoelectric focusing can be performed using the following step transformation profile.

20 minutes at 200V, 15 minutes at 450V, 15 minutes at 750V, 30 minutes at 2000V.

Constant power supply (e.g., PowerEase (registered ^{trademark) 500 (PowerEase (R) 500} ) (Invitrogen Corp., Carlsbad, CA) to achieve the isoelectric focusing superior results by using the following with reference to Even can.

3-4 hours at 500V Subject is 7cm IPG strip.

  Therefore, using the cassette and buffer core of the present invention, IPG isoelectric focusing can be performed using a maximum voltage of only 3500 V, 3000 V, 2750 V, 2500 V, 2250 V, 2000 V, 1750 V, 1500 V, 1250 V, 1000 V, only 750 V or 500 V. IPG isoelectric focusing can be performed using a stepless, stepped, or constant voltage profile. The minimum voltage may be 500V, 750V, 1000V, 1250V, 1500V, 1750V, 2000V, 2250V, 2500V or more.

  In general, the electrical parameters required for effective isoelectric focusing using an IPG strip are said to be the minimum or desired number of volts-time. Here, volt-time is defined as the cumulative sum of voltage × time for each stage of the profile. As mentioned above, the nominal voltage reported by the power supply is always greater than the voltage actually applied to the strip, and the nominal volt-time to actual volt-time ratio depends on the efficiency of the device.

  In general, prior art approaches to IEF using IPG strips require at least 13,000 nominal volt-hours and, depending on the separation, 36,000 nominal volt-hours.

  Depending on the increased efficiency of the cassette and buffer core of the present invention, 70 mm, 80 mm, 90 mm, 100 mm, 110 mm, 120 mm, 120 mm, 130 mm, 140 mm, 150 mm, 160 mm, 170 mm, 180 mm, 190 mm, 200 mm, 210 mm, 220 mm, 240 mm, or For strips of 240 mm and shorter, longer, or intermediate lengths, less than 13,000 nominal volts-hour, generally 12,000 nominal volts-hour, 11,000 volts, 10, 000th bolt time, 9000th bolt time, 8000th bolt time, 7000th bolt-hour, 6000th bolt-hour, 5000th bolt-hour, 4000th bolt-hour, 3000th bolt-hour, Either 2000,1900,1800,1700,1600,1500,1400 or slightly 1300,1200,1100 or 1000 nominal volts, - we are possible to realize an isoelectric less than time.

  Thus 70 mm, 80 mm, 90 mm, 100 mm, 110 mm, 120 mm, 120 mm, 130 mm, 140 mm, 150 mm, 160 mm, 170 mm, 180 mm, 190 mm, 200 mm, 210 mm, 220 mm, 240 mm, or 240 mm, and shorter or longer, and For medium length gel strips, 1000-12,000 nominal volt-hours, 1100-11,000 nominal volt-hours, 1200-10,000 nominal volt-hours, 1300-9000 nominal volt-hours, Isoelectric focusing can be achieved after 1400-8000 nominal volt-hours and 1500-7000 nominal volt-hours of electrophoresis.

  As is well known in the art, the optimal voltage profile and time will vary from sample to sample and will depend on other variables known in the art such as power source, number of strips simultaneously isoelectric focusing, and pH range of IPG strips. Can change. For example, pH 6-10 IPG strips may require isoelectric focusing for an extra 30 minutes. Similarly, samples containing crude protein mixtures or high salt concentrations (eg,> 10 mM) may require longer run times or total volt times for optimal resolution. Starting from the above guidelines, it is well known in the art to empirically optimize isoelectric focusing conditions by routine experimentation, for example, by changing any one or more of voltage, time, and profile. Within the scope of the technology.

  The increased electrical efficiency of the cassette and buffer core of the present invention allows the use of lower voltages and more efficient currents, reducing the arcing and burning tendency of IPG strips.

  When electrophoresis is complete, the buffer core 26 with the cassette 100 may be stored in a sealed container at −80 ° C. until the strip is ready for analysis.

  The strip 20 may be withdrawn from the cavity 18 for subsequent processing, with or without prior refrigeration, generally. As already described, the strip 20 may be removed through the channel inlets 14 and 16, sometimes after drying, but the strip 20 is generally removed by enlarging the dimensions of the cavity 18 in the channel 12. In the multi-layer laminate embodiment of the cassette 100, this is done by separating the laminate cover 42 from the shape retention member 10.

  Channel inlets 14 and 16 for each of the channels present in the buffer core allow electrical connection with the channel cavity 18 therebetween, for example through the common surface of cassette 100 as shown in FIGS. The buffer core embodiments described above are designed to facilitate cassette electrophoresis.

  However, such a geometric arrangement is not essential. Accordingly, the present invention further provides a cassette in which the inlets 14 and 16 do not open on the same side of the shape retaining member 10 and a buffer core suitable for electrophoresis of such a cassette. A major advantage of such a geometrical arrangement is that the cassette can be made compatible with the buffer cores currently sold for slab gel SDS-polyacrylamide gel electrophoresis.

  FIG. 7A is a front view and FIG. 7B is a side view of the cassette 1000 of the present invention where the inlets 114 and 116 of the channel 112 each open to the opposite side of the cassette 1000.

  The dimensions of the channel 112 of the cassette 1000 are set to removably engage the precast hydratable electrophoretic separation medium in a dehydrated state and to accommodate the strip after rehydration, similar to the channel 12 of the cassette 100. Is done.

  Of course, in order to perform electrophoresis using the cassette 1000 as an encapsulating member, the cathode and anode must establish an electrical connection with the strip 20 from opposite sides of the cassette 1000.

  As when the inlets 14 and 16 open the channel 12 on the same side of the cassette 100, the electrical connection of the channel 112 through the inlets 114 and 116 may also be direct, such as by passage of the electrodes through the respective inlets. Alternatively, it may be indirect, such as through mediation of polymer gels and / or conductive wicks. However, when the inlets 114 and 116 are opened on opposite sides of the cassette 1000, the electrical contacts are made individually by contact of the anode and cathode electrodes with the first buffer reservoir and the second buffer reservoir. Contact may be established and the sump now contacts inlets 114 and 116 individually.

  In the latter case, the first and second buffer reservoirs must be kept electrically isolated from each other, except for the circuit completed via the separation medium of the strip 20.

As further described in commonly assigned US Pat. No. 5,888,369, which is hereby incorporated by reference in its entirety, and as commercially available from Invitrogen Corporation, a plurality of cassettes 1000, Alternatively, such a geometry can be easily implemented by sealingly contacting a single cassette 1000 and buffer dam with the buffer core 126. (XCell II ^™ Buffer Core with electrodes, catalog number EI9014X, Invitrogen Corp., Carlsbad, CA).

  To perform electrophoresis using such a system, as shown in FIGS. 7C and 7D, two cassettes 1000 (or a single cassette 1000 and a buffer dam) are positioned in use with a buffer core 126. Fit together and house.

  The assembled buffer core and cassette are then engaged within the electrophoresis buffer chamber 34. The buffer chamber by itself or in conjunction with another device places a plurality of cassettes 1000 (or a single cassette 1000 and a buffer dam) in sealable contact with the buffer core 126. Preferably, such another device may be a cam activated clamp such as that further described in US Pat. No. 6,001,233, which is hereby incorporated by reference in its entirety. Alternatively, buffer core 126 may first be gently engaged within electrophoresis buffer chamber 34, after which the cassette is aligned with buffer core 126, brought into contact, and then further pressed.

  In general, although necessary, a liquid-tight contact between the buffer core 126 and the cassette 1000 (or buffer dam) is further facilitated by a gasket fitted into the groove 170 of the buffer core 126, such as a silicone gasket.

  A buffer core 126 and a plurality of cassettes 1000 (or a single cassette 1000 and a buffer dam) engaged with the buffer core define the shape of the internal chamber 162. If the internal chamber 162 does not overflow the cassette 1000, it is not connected to the electrophoresis buffer chamber 34 by a fluid. The inner chamber 162 is then contacted with (i) a cassette inlet 114 (or 116 in some cases) that opens into the chamber 162 and (ii) is added to a level that does not overflow the cassette 1000. In addition, a conductive aqueous solution is (i) brought into contact with the cassette inlet 116 (or 114 in some cases) that opens into the electrophoresis buffer chamber 34, and (ii) is added to a level that does not overflow into the cassette 1000.

As further described in commonly assigned US Pat. No. 5,888,369, and as known to users of the X Cell ^™ SureLock system, the geometry of the electrodes of the buffer core 126 The anode contacts the internal chamber 126 and the cathode contacts the external reservoir 60 formed in the chamber 34, thus allowing the voltage gradient necessary to achieve electrophoresis to be applied to the strip 20.

  It should be noted that a potential problem with direct contact between the channel 112 and strip 20 and the liquid reservoir is an increased possibility of leakage of the ampholyte and / or sample from the separation medium.

  As used herein, the cassette of the present invention as having at least one preformed channel with sufficient dimensional stability to allow hydration accommodation of a precast hydratable separation medium engaged therein. As described above in detail, the pre-formed channel is only one way to hydrate and contain such media in the encapsulating member.

  As an example only, if the encapsulating member is malleable but retains shape, the encapsulating member is wrapped around the strip in a dehydrated form to form a new channel and rehydrated once the strip is hydrated The strip may be accommodated inside and sealed.

  In yet another aspect, the present invention provides kits that facilitate the performance of the methods of the invention.

  The kit of the present invention may comprise at least one component (single or plural) selected from the group consisting of: Buffer core of the present invention, electrophoresis chamber, electrophoresis chamber lid capable of establishing electrical contact with the buffer core, tension wedge, buffer dam, electrode wick, seal tape, IPG strip, container-filled carrier ampholyte, container-filled cathode Buffers, anodic buffers in containers, stained dyes in containers (eg, silver stain or colloid blue stain), protein standard samples, typically analyte standard samples of mixtures, and polyacrylamide gels suitable for performing two-dimensional separations .

  Thus, a first series of kit embodiments may include non-disposable items useful in adapting the electrophoresis apparatus to receive the cassette of the present invention. For example, a kit may include a buffer core of the present invention and a corresponding lid.

  For users who do not also have a tension wedge on the electrophoresis chamber, for example, a cam-activated tension wedge suitable for applying pressure to press the cassette 100 inward against the buffer core, the kit of the present invention provides an electrophoresis chamber, tension wedge, May include a buffer core and a buffer lid. Preferably, the kit further includes a buffer dam as required to allow execution on a single cassette.

  A second series of kit embodiments may include disposable items suitable for use with the present invention.

  For example, the kit may include at least one cassette and a plurality of electrode wicks. If desired, the kit may further include a sealing tape suitable for sealing inlets 14 and 16 during rehydration and / or either the same pH range or a different pH range as required. May include a plurality of IPG strips.

  If necessary, the kit of the present invention can also be used with any one or more carrier ampholytes, cathode buffers, anode buffers, dyes (such as silver dyes or colloid blue dyes) in individual containers—use Either in a concentrated liquid form for dilution at a concentration of 1 or later, or in a dry form for reconstitution with appropriate quality water-protein standard (generally a mixture), or isoelectric focusing Polyacrylamide gels such as Tris-Glycine ZOOM® gel (Invitrogen Corp., Carlsbad, Calif.) Suitable for performing two-dimensional separation following electrophoresis may be included.

Example 1
Determination of Channel Allowance Three cassettes having essentially the geometric shape shown in FIG. 1A were produced by machining each of six parallel channels in a shape-retaining plastic slab. The six channels of the first cassette are all 0.77 mm deep, the two channels are 4.09 mm wide, the two channels are 0.65 mm wide, and the two channels are 3.35 mm wide. It was. The six channels of the second cassette are all 0.65 mm deep, the two channels are 4.09 mm wide, the two channels are 0.65 mm wide, and the two channels are 3.35 mm wide. It was. The six channels of the third cassette are all 0.57 mm deep, the two channels are 4.09 mm wide, the two channels are 0.65 mm wide, and the two channels are 3.35 mm wide. It was. By applying a flexible laminate cover to each of the three cassettes, the channel was made fluid-tight with the exception of the end inlet.

  5 μL of Serva IEF standard solution (Catalog No. 39212-01, Serva Electrophoresis GmbH, Heidelberg, Germany) was mixed with 120 μL of rehydration buffer having the following composition. 8.0 M urea, 0.5% ampholyte (3-10 IPG buffer, catalog number 17-6001-11, Amersham Biosciences), 2.0% (weight / volume) CHAPS, 20 mM DTT, 0.0025% ( (Weight / volume) Bromophenol blue. The cassette was placed horizontally and the aqueous solution was pipetted into each channel of the three cassettes.

  Immobiline DryStripe 3-10 7 cm gel (Amersham Biosciences, Piscataway, NJ, USA) was inserted into each channel. The channel inlet was covered with cover tape and the strip was rehydrated for 8 hours. The cover tape was removed and the sample introduction well was removed, if any.

  At the end entrance, a filter paper wick moistened with water was placed in contact with the extreme end of the gel portion of the strip.

  Electrodes were contacted with the wick at the anode and cathode ends of the cassette, and voltage was applied in three steps according to the following protocol. 15 minutes at 250 volts, 1 hour 30 minutes at 250 to 3500 volts continuously variable transformer, 1 hour at 3500 volts. In all stages, the current was limited to 1 mA and the power was limited to 4 watts.

  Strips were removed, stained with Coomassie Blue stain, aligned and photographed.

  The results shown in FIG. 8 show that the proper channel of the Serva IEF standard (leftmost lane) in a strip having a nominal width of 3 mm and a depth of 0.5 mm, even for the largest channel with a width of 4.09 mm and a depth of 0.77 mm. It shows that electric point electrophoresis is possible.

Example 2
Comparison of Electrical Properties Using a Stepless Transform Profile This experiment used a stepless transformer power profile to (i) a commercially available horizontal IPG electrophoresis apparatus (Multiphor ^™ flatbed system, Amersham Biosciences, Inc. , Piscataway, NJ) and (ii) the actual current flowing in the IPG strip in the cassette of the invention using the buffer core of the invention in vertical electrophoresis (ZOOM® IPGRunner ^™ ). Compare quantities. For further comparison, strips from two vendors (Amersham Biosciences, Inc. “AP Strip” and Invitrogen Corporation “INV Strip”) were used.

Equipment and materials Power supply Novex Model 35-40. Four of these devices were used, one for each of four individual experimental devices.

a. Voltage / time profile The same stepless transformation profile was set for each of the four systems. The profile settings were as follows: 0 → 250V in 15 minutes
250 → 3500V in 1.5 hours
2 hours at 3500V Digital multimeter (DMM) for measurement.

  In both cases, two groups capable of measuring microamperes were used. DMM was used in the current measurement mode. The DMM was connected in series with the power cable on the ground side between the device and the power source.

a. Fluke 87 series III
b. Fluke 73 Series III
3. Multiphor ^TM
a. Wick liquid 0.5ml ultrapure water.

b. Rehydration 0.125 ml / strip 4. ZOOM (registered trademark) IPG Runner ^TM
a. Wick solution 0.75 ml deionized water b. Rehydration 0.155 ml / strip 5. Strip a. Invitrogen 4-7 pH, lot number 100248
b. AP Biotech 4-7 pH, lot number 287374
6). Sample in rehydration solution a. Second centrifugal lysate from E. coli (0.002 ml per ml of rehydration solution)
b. Centrifugation at 14,000 rpm for 15 minutes after addition of lysate c. pH 4-7 ZOOMlytes (Invitrogen Corp.)
Results table 1
Real current when using Multiphor ^TM and ZOOM® IPG Runner ^TM

Footnotes in Table 1
¹ Burned at the acid end of one strip after 10 minutes at 3500V. The current dropped again after burning for 5 minutes.

² Burning current / Afterburning current. Burning started with one strip after 3 hours at 3500V. Burning lasted 7 minutes. Since the power limit of the power supply was reached, the voltage dropped when the current spiked during burning. For example, the voltage was 2900 at 0.65 mA. The highest current recorded (100 millisecond sampling) was 0.82 mA during burning.

³ one hour 40 minutes of 3500V phase of 2 hours was measured current.

  The results are plotted in FIG. As can be seen from Table 1 and FIG. 9, when using the cassette and buffer core of the present invention compared to a flat bed type device, both types of IPG strips have substantially higher currents throughout the experiment. There is a flow.

  Table 2 shows the “volt-time” ratio during the 3500 V fixed period of the power cycle program for these two types of devices.

The “volt-time” ratio is calculated from the measured current—assuming the resistance in the IPG strip is the same, the measured current-time ratio is equal to the volt-time ratio. The ratio is calculated as IPG Runner ^™ (Volt-Time) divided by Multiphor ^™ (MP) “Volt-Time”.

Table 2
"Volt-time" ratio

Table 2 is apparently the same in nominal volt-time calculated by multiplying the power supply voltage by time, but the cassette and buffer core of the present invention are flatbed multiphor in terms of transferring power to the gel. ^It is much more efficient than ^TM .

During the first low voltage phase of the profile, the cassette and buffer core of the present invention pushes the amount of current in the strip (both AP strip and INV strip) up to four times that of Multiphor ^™ . There was a visible difference in the speed and shape of the dye tip between the two devices during this stage.

In this experiment, during the high voltage isoelectric focusing plateau set at 3500 V, the cassettes and buffer cores of the present invention averaged about 2.6 times the Multiphor ^™ and AP, respectively, and the INV strip. Provided 2.9 times more current.

  Isoelectric focusing may be achieved with the cassettes and buffer cores of the present invention using a continuously variable transformer profile and using less voltage and time than used for conventional flatbed devices. For shorter periods, lower voltages are more convenient, and less sophisticated (and less expensive) power supplies are sufficient, do not require current limit power supplies, and less frequent strip arcing and therefore burning Should be connected. The optimum voltage and time depend in part on the sample osmolarity and electrolyte concentration, as in the previous examples.

Example 3
Electrical properties using a stepped voltage profile To evaluate the effectiveness of a voltage step profile without current or watt limits, each standard strip is loaded with strips of various pH ranges and six strips of limited capacity are used. The four cassettes housed were run in two ZOOM® IPG Runner ^™ systems (cassette, buffer core, tank) of the present invention.

(Equipment and materials)
1. Power supply Novex Model 35-40. Two of these devices were used, one for each of two separate experimental devices.

  a. Voltage / Time Profile As shown in Table 3 below, the same voltage profile was set for both systems.

  2. Digital multimeter for measurement. In both cases, two groups capable of measuring microamperes were used. DMM was used in the current measurement mode. The DMM was connected in series with the power cable on the ground side between the device and the power source.

a. Fluke 87 series III
b. Fluke 73 Series III
Table 3
Step voltage profile

Results The results are summarized in Table 4 and plotted in FIG.

Table 4
Measurement current and nominal voltage, stair profile

Footnotes in Table 4
No ^one strip burning.

  In the step voltage profile, no burning occurred in any of the 24 strips.

  At the 500 volt step, it was 99 microamperes per strip in less than 1 minute, but was above 70 microamperes for about 5 minutes. However, 500 volts is much lower than the final isoelectric focusing voltage, and it is unlikely that the conductivity of such a low voltage sample will cause arcing or burning. Neither arcing nor burning was seen.

This experiment was performed using a variety of IPG strips with the cassette and buffer core of the present invention (ZOOM® IPG Runner ^™ system) by means of an inexpensive power supply without adjustment circuitry that can only be a “step” profile. This demonstrates that samples can be subjected to isoelectric focusing with high quality isoelectric focusing without burning. The power supply is programmable but does not provide current limiting in the microampere range.

  Furthermore, the tested profiles appear to provide a very good balance by placing a higher current load in the lower voltage range. However, the optimum voltage and time depend in part on the sample osmolarity and electrolyte concentration.

  All patents and publications cited herein are hereby incorporated by reference as if each were specifically and individually incorporated herein by reference. Although the invention has been described in some detail by way of illustration and example, the appended claims, which uniquely define the scope of the invention, together with the equivalents of the claims over the full scope, are taught according to the teachings herein. It will be readily apparent to those skilled in the art that changes and modifications can be made therein without departing from the spirit or scope of the invention.

These and other objects and advantages of the present invention will become apparent from the detailed description considered in conjunction with the accompanying drawings. The same letters refer to the same parts throughout the drawings.
FIG. 1A is a front perspective view of one embodiment of the cassette of the present invention. FIG. 1B is a front perspective view of another embodiment of the cassette of the present invention. 1C is a front perspective view of the opaqued embodiment of FIG. 1B. FIG. 2 is a front perspective view of the cassette of the present invention with an IPG strip inserted into one of the six available channels. FIG. 3A is a front perspective view of the cassette of the present invention before the conductive wick is mounted with the well forming member removed. FIG. 3B shows that the first conductive wick contacts the anode ends of the three IPG strips present in the six available channels and the second conductive wick contacts the cathode ends of the three IPG strips. It is the front perspective view of the cassette of this invention which is doing. FIG. 3C is a rear perspective view of an embodiment of the cassette of the present invention, particularly showing indented areas that facilitate heat dissipation during electrophoresis. FIG. 3D is a rear perspective view of another embodiment of the cassette of the present invention specifically illustrating a plurality of indented areas that facilitate heat dissipation during electrophoresis. FIG. 4 is a side exploded perspective view of the multilayer laminate cassette of the present invention. FIG. 5 is a side exploded perspective view of the document injection well assembly of the cassette of the present invention. FIG. 6A is a front perspective view of a buffer core of the present invention in which neither an anode wiring nor a cathode wiring is mounted and a gasket is mounted. FIG. 6B is a front perspective view of the buffer core of the present invention (front) in which the anode and cathode electrodes are aligned in use so that they are in contact with the anode and cathode wick (rear) of the cassette of the present invention, respectively. 6C is a front perspective view of the buffer core and cassette of FIG. 6B in contact with each other when in use. FIG. 6D is a front perspective view of the buffer core in contact with the two cassettes of the present invention. FIG. 6E shows the buffer core of the present invention with the cassette of the present invention engaged in use, and further engaged within the electrophoresis chamber; FIG. 7A is a front view of the cassette of the present invention with the channel inlet open to the opposite side of the cassette. FIG. 7B is a side view of the cassette of FIG. 7A. FIG. 7C is an exploded perspective view of the two cassettes shown in FIGS. 7A and 7B. Shows the relationship when using the buffer core of the prior art, FIG. 7D is a perspective view of the cassette of FIGS. 7A and 7B in contact with a prior art buffer core. FIG. 8 shows the IPG strip after electrophoresis in a channel with the indicated internal dimensions. FIG. 9 shows the measured currents in the IPG strip flowing in the cassette of the present invention and in the commercial horizontal flatbed apparatus using the buffer core of the present invention with the same voltage program using the same power source for comparison. It is a graph to plot. In FIG. 10, isoelectric focusing was performed with a stepped voltage profile using the cassette and buffer core of the present invention to plot the current measured in the IPG strip and the voltage reported by the two power supplies. It is a graph to do. FIG. 11 illustrates an electrophoresis tank cover useful for applying a voltage to the cassette and buffer core of the present invention.

Claims (35)

A system for low resistance electrophoresis of an analyte sample in a precast hydratable separation media strip comprising:
Means for enclosing a plurality of said strips, wherein said enclosing means permitting spaced electrical contact separately from each said encapsulated strip through respective first and second inlets; and external compression force Means for simultaneously realizing electrical contact with a spacing between each of the enclosed strips by a single anode and a single cathode in response to
The electrical contact means distributes an external compressive force to cause the anode and the cathode to be at the first and second inlets at a greater pressure than elsewhere on the encapsulation means. A system that can be pressed against the encapsulation means. The system of claim 1, wherein the encapsulating means is capable of containing the strip therein for hydration. The electrical contact means is an anode support,
Cathode support,
3. A system according to claim 1 or claim 2, comprising an anode and a cathode, wherein the support discontinuously distributes external compressive force to the anode and cathode, the first and second inlets. A system for pressing the anode and the cathode against the encapsulating means with a greater pressure than elsewhere on the enclosing means. The system of claim 2, wherein the anode support contacts the anode discontinuously and the cathode support contacts the cathode discontinuously. The system of claim 1, wherein electrophoretic separation in the strip is possible with an application of 3000 volts or less. The system of claim 5, wherein electrophoretic separation in the strip is possible with an application of 1500 volts or less. The system of claim 6, wherein electrophoretic separation in the strip can be achieved with an application of 500 volts or less. The system of claim 1, wherein electrophoretic separation in the strip is possible with an application of less than 2000 volts-hour. 9. The system of claim 8, wherein electrophoretic separation in the strip can be achieved with an application of less than 1500 volts-hour. The system of claim 1, wherein electrophoretic separation in the strip can be accomplished in less than 6 hours. 11. The system of claim 10, wherein electrophoretic separation in the strip can be done in less than 5 hours. 11. The system of claim 10, wherein electrophoretic separation in the strip can be done in less than 4 hours. A method for low resistance electrophoresis of an analyte sample in a precast hydratable separation media strip comprising:
Housing and hydrating at least one strip in an encapsulating member that allows for electrical contact with each of a plurality of encapsulated strips through respective first and second inlets;
Applying a sample containing a protein analyte to the at least one strip;
Forcibly pressing the anode and cathode against the encapsulating member to achieve electrical contact with simultaneous spacing with each of the strips, and distributing the force pressing the anode and cathode against the encapsulating member Creating a greater contact pressure at the first and second inlets than elsewhere on the encapsulating member; and
Applying a potential to the anode and the cathode with a potential difference and time sufficient to achieve electrophoretic separation of the analyte in the at least one strip;
Including methods. The method of claim 13, wherein the sample is applied during containment of the strip in the encapsulating member. The method of claim 13, wherein the applied potential difference is 3000 volts or less. The method of claim 15, wherein the applied potential difference is 1500 volts or less. The method of claim 16, wherein the applied potential difference is 500 volts or less. 14. The method of claim 13, wherein the potential difference is applied for less than 6 hours. The method of claim 18, wherein the potential difference is applied for less than 5 hours. The method of claim 19, wherein the potential difference is applied for less than 4 hours. 21. The method of claim 20, wherein the potential difference is applied for less than 3 hours. 14. The method of claim 13, wherein the potential difference is applied less than 2000 volts-hour. 24. The method of claim 22, wherein the potential difference is applied less than 1500 volts-hour. 24. The method of claim 23, wherein the potential difference is applied less than 1400 volts-hour. The method of claim 13, wherein the potential difference is applied in multiple ramp voltage steps. The method of claim 13, wherein the potential difference is applied in a plurality of stepped voltage steps. The method of claim 13, wherein the potential difference is applied at a constant voltage level. 27. A method according to any one of claims 13 to 26, wherein the strip is an IPG strip. A plurality of precast hydratable separation media strips encapsulated in a means for pressing the anode and cathode and individually allowing electrical contact with each of the encapsulated strips through the first and second inlets, respectively And a device for forcing electrical contact with simultaneous spacing, the following:
A substantially flexible frame;
An anode support;
Cathode support;
An anode; and a cathode;
The anode support and the cathode support are spaced apart from the frame, and an external compressive force is distributed to the cathode and the anode, respectively, and the first and second inlets An apparatus capable of pressing the cathode and the anode against the strip with a higher contact pressure than elsewhere on the enclosing means. 30. The apparatus of claim 28, wherein the anode support is in intermittent contact with the anode and the cathode support is in intermittent contact with the cathode. In an electrophoresis method using a precast hydratable separation media strip, the following:
Contacting the strip with the anode and cathode in spaced contact with a sufficiently low resistance between the gel and the power supply to allow electrophoretic separation at a maximum applied voltage of 3000 volts or less;
A method comprising an improvement comprising: 32. The method of claim 31, wherein the highest applied voltage is 1500 volts or less. The method of claim 32, wherein the highest applied voltage is 500 volts or less. 32. The method of claim 31, wherein the strip is an immobilized pH gradient (IPG) strip and the resistance is low enough to allow isoelectric focusing with an application of nominally less than 2000 volts-hour. 35. The method of claim 34, wherein the resistance is low enough to allow isoelectric focusing with an application of nominal less than 1500 volts-hour.

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