WO2003106973A2 - Methods and apparatus for low resistance electrophoresis of prior-cast, hydratable separation media - Google Patents
Methods and apparatus for low resistance electrophoresis of prior-cast, hydratable separation media Download PDFInfo
- Publication number
- WO2003106973A2 WO2003106973A2 PCT/US2003/019335 US0319335W WO03106973A2 WO 2003106973 A2 WO2003106973 A2 WO 2003106973A2 US 0319335 W US0319335 W US 0319335W WO 03106973 A2 WO03106973 A2 WO 03106973A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- strips
- anode
- cathode
- strip
- cassette
- Prior art date
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44795—Isoelectric focusing
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
Definitions
- the present invention relates to methods and apparatus for electrophoresis of prior- cast hydratable separation media.
- the invention relates to methods, cassettes, buffer cores, and systems useful for conducting isoelectric focusing using immobilized pH gradient (IPG) strips.
- IPG immobilized pH gradient
- isoelectric focusing has served as a primary tool for analyzing proteins present in complex admixture, such as proteins present in biological samples.
- proteins are driven by an applied electric field through a pH gradient typically established in a support matrix, such as a gel. Proteins migrate until the isoelectric point (pi) of the protein coincides with the local pH; at that point, the protein no longer bears net charge and ceases to migrate, becoming focused at a point that is characteristic of the protein.
- the pH gradient for IEF was established and sustained in the gel matrix by mobile carrier a pholytes (CA).
- Gels typically would be polymerized in the presence of a population of CA having a range of charge characteristics; upon application of a voltage gradient, the various species of CA would align themselves in the matrix to establish a pH gradient across the gel.
- Cathodic drift can be reduced by casting IEF gels in enclosed tubes, thus limiting exposure to atmospheric CO2.
- the tube traps prepolymer component impurities in the matrix during polymerization, interfering with separation.
- the tube format presents difficulties when a second dimension of separation, such as fractionation by size, is desired.
- IPG immobilized pH gradient
- IPG not only reduced the problem of cathodic drift, but also proved useful in reducing interference from prepolymer component impurities, since the IPG strip's plastic backing imparts sufficient structural resilience to the gel as to permit the gel to be washed before use. The increased resilience also permits the gels to be stored in dehydrated form before use.
- Dehydrated IPG strips are today sold in a variety of pH ranges and a variety of separation lengths by a number of vendors (e.g., Immobiline DryStrip Gels, Amersham Biosciences, Piscataway, NJ, USA; ReadyStrip IPG, Bio-Rad Laboratories, Hercules, CA, USA).
- a continuous pKa gradient is immobilized on at least one of the major opposing surfaces of a cavity formed between two plates.
- the cavity which can be further segmented into parallel channels, is then filled with a flowable separation medium.
- Electrophoresis is preferably conducted with the assembly oriented horizontally to minimize convection currents in the flowable separation medium.
- the apparatus does not readily permit insertion of prior-cast hydratable separation media, such as commercial IPG strips, nor does it readily permit electrophoresis in the vertical dimension.
- the present invention solves these and other needs in the art by providing methods, apparatus, and kits for electrophoresis of prior-cast hydratable separation media that obviate the use of an occlusive oil layer, thereby obviating the requirement that electrophoresis be 5 performed in the horizontal orientation.
- the present invention is based, in part, upon the discovery that the swelling that attends rehydration of prior-cast hydratable separation media can be exploited to help lodge such media in an enclosure that permits spaced electrical communication with the enclosed separation medium.
- the spaced electrical communication makes it possible to apply a voltage o gradient to the prior-cast hydratable separation media while the medium is otherwise enclosed, permitting electrophoresis to be conducted within a cassette.
- the separation medium's contact with air is substantially reduced.
- the reduction in air contact obviates the prior art requirement for occlusive contact with a fluid oil layer during 5 immobilized pH gradient isoelectric focusing.
- the prior-cast separation medium can be electrophoresed in any physical orientation.
- the prior-cast hydratable separation medium is an IPG strip
- relaxation of the prior-art requirement for horizontal electrophoresis makes it newly possible to perform IPG electrophoresis using the widely o distributed, small footprint, vertical electrophoresis gel boxes presently used to perform SDS-
- the invention is further based upon novel apparatus designs that minimize the resistance between power supply and gel; the reduction in parasitic system impedances permits separations, particularly isoelectric focusing in iPG strips, to be performed using lower 5 voltages for reduced times .
- the invention provides a method for performing electrophoresis, comprising: hydratingly lodging a prior-cast hydratable electrophoretic separation medium within an enclosing member that permits spaced electrical communication with the enclosed medium; and then using the spaced electrical communication to establish a voltage gradient in o the enclosed separation medium sufficient to effect electrophoretic separation of analytes therein.
- the method further comprises the antecedent step of inserting the prior-cast hydratable electrophoretic separation medium in its dehydrated state into the enclosing member.
- the method further includes a later step of removing the prior-cast hydratable electrophoretic separation medium from the enclosing 5 member.
- the medium once removed can be used, for example, to apply the one- dimensionally fractionated sample to a gel to effect a second dimension of separation.
- the step of hydratingly lodging comprises: contacting the dehydrated prior-cast hydratable electrophoretic separation medium with an aqueous solution, often an aqueous solution that includes the sample to be fractionated. o
- the methods of the present invention are particularly useful in performing isoelectric focusing using immobilized pH gradient strips.
- the prior-cast hydratable electrophoretic separation medium used in the practice of the present invention can usefully have an immobilized pH gradient.
- the methods of the present invention include the use of an 5 enclosing member that has (i) means for hydratingly lodging a prior-cast electrophoretic separation medium therewithin, and (ii) means for spaced electrical communication with the enclosed separation medium, wherein the spaced electrical communication means can be used to apply a voltage gradient to the enclosed medium sufficient to effect electrophoretic separation of analytes present therewithin.
- the invention provides a cassette for performing electrophoresis, comprising: means for hydratingly lodging a prior-cast electrophoretic separation medium within an enclosing member; and means for spaced electrical communication with the enclosed medium, wherein the spaced electrical communication means can be used to establish a voltage gradient in the separation medium sufficient to effect 5 electrophoretic separation of analytes therein.
- the cassette of the present invention comprises: a form- retaining member, and at least one channel, wherein the form-retaining member imparts dimensional integrity to the channel or channel(s).
- the cassette includes a plurality of such channels.
- the form-retaining member contributes the entire circumferential wall of the cavities of the channels.
- the cassette further comprises a laminate cover; the laminate cover adheres directly or indirectly to the form-retaining member and contributes at least part of the circumferential wall of said channels. In these latter embodiments, the adherence of the laminate cover to the form- o retaining member is typically reversible.
- the cassette further comprises a first well-forming member, which adheres directly or indirectly to the form-retaining member, and which defines fluid reservoirs at a plurality of first channel entries.
- the cassette can further comprise a second well-forming member, the second well-forming member adhering, directly or indirectly, 5 to the form-retaining member and defining fluid reservoirs at a plurality of second channel entries.
- the well-forming members can usefully be reversibly adherent to the form-retaining member.
- the first and second channel entries for each of the channels permit electrical communication with the intervening channel cavity through a o common surface of the cassette. In another series of related embodiments, the first and second channel entries permit electrical communication with their intervening cavity through separate surfaces of the cassette. These two mutually exclusive geometries call for different electrode geometries, and thus different electrophoresis buffer cores, to complete the circuits required for electrophoresis. 5 [0032]
- the prior-cast hydratable electrophoretic separation medium can be provided by the user, can be included within one or more channels of the cassette without requirement for user insertion thereof, or can be provided separately packaged with the cassette in a kit.
- kits for facilitating electrophoresis of prior-cast hydratable electrophoretic separation media typically comprise a cassette of the present invention and at least one prior-cast hydratable electrophoretic separation medium suitably dimensioned as to be hydratingly lodgeable in said cassette.
- the kit includes a cassette and at least one conductive wick for use therewith; often, in such kits, a sufficient number of wicks are provided to facilitate both anodic and cathodic connections with the cassette.
- the cassettes of the present invention can be used to effect vertical electrophoresis of 5 prior-cast hydratable separation media, usefully in the buffer tanks that are commonly used, with buffer cores, for SDS-PAGE electrophoresis.
- buffer cores presently used for SDS-PAGE electrophoresis can be used.
- alternative buffer core o geometries are required.
- a buffer core for vertical electrophoresis of pre-cast hydratable electrophoretic separation media comprising: a substantially inflexible frame, an anode, and a cathode in spaced relationship to the anode.
- the buffer core frame has a first cassette engagement face and a second cassette 5 engagement face. Operational engagement of a first and second cassette to the respective first and second frame engagement faces creates a chamber internal to the frame that is sealed on 5 sides.
- the cathode and anode are each in electrical communication with the interior of the internal chamber, and operational engagement of a first and second cassette to the respective first and second frame engagement faces causes spaced contact of the anode o and cathode to the surface of at least one cassette that engages the frame engagement surface, allowing electrophoresis of prior-cast hydratable separation media enclosed therein.
- the cassette and buffer core system of the present invention reduces the resistance between power supply and gel, permitting electrophoretic separation using lower voltages, for shorter times, for a lower volt-hour total. 5 [0038]
- the invention provides a system for low resistance electrophoresis of analyte samples in prior-cast, hydratable separation media strips.
- the system comprises means for enclosing a plurality of strips and means responsive to an external compressive force for effecting spaced electrical communication by a single anode and single cathode simultaneously with each of said enclosed strips.
- the enclosing means permits spaced electrical communication separately with each of the enclosed strips through respective first and second entries.
- the electrical communication means is capable of distributing an external compressive force to urge the anode and the cathode toward the enclosing means with greater pressure at the first and second entries than elsewhere on the enclosing means.
- the electrical communication means may comprise an anode support; a cathode support; an anode; and a cathode.
- the supports in these embodiments discontinuously 5 distribute an external compressive force to the anode and cathode to urge the anode and cathode toward the enclosing means with greater pressure at the first and second entries than elsewhere on the enclosing means.
- the anode support makes discontinuous contact with the anode and the cathode support makes discontinuous contact with the cathode.
- the enclosing means is capable of hydratingly lodging strips there within.
- the system provides a substantially reduced resistance pathway between power supply and gel than do prior art devices for electrophoresis of prior-cast, hydratable, separation media, such as IPG strips.
- the system of this aspect of the invention may, for example, be capable of effecting electrophoretic separation, including isoelectric focusing in IPG strips, with application of a maximum of 3000 or fewer volts, 1500 or fewer volts, even 500 or fewer volts.
- the system may be capable of effecting electrophoretic separation, including isoelectric focusing in IPG strips, with application of fewer than 2000 volt-hours, even as few as 1500 volt-hours.
- the o system of this aspect of the invention may be capable of effecting electrophoretic separation, including isoelectric focusing in IPG strips, in fewer than 6 hours, 5 hours, even 4 hours or less.
- the invention provides a method for low resistance electrophoresis of analyte samples in prior-cast, hydratable separation media strips.
- the method comprises hydratingly lodging at least one strip within an enclosing 5 member that permits separate, spaced, electrical communication with each of a plurality of enclosed strips through respective first and second entries; applying a sample containing protein analytes to the enclosed strip; forcibly urging an anode and a cathode toward the enclosing member to effect simultaneous spaced electrical communication with each of the enclosed strips, wherein the force urging the anode and the cathode toward the enclosing o member is distributed to create greater contact pressure at the first and second entries than elsewhere on the enclosing member; and then applying electrical potentials to the anode and cathode at a potential difference and for a time sufficient to effect electrophoretic separation of analytes in the enclosed strips.
- sample is applied during lodging of the strip in said enclosing member. 5 [0047] The method provides a substantially lower resistance pathway between power supply and separation media than is found in the prior art.
- effective separation including isoelectric focusing in IPG strips
- the methods may effect electrophoretic o separation, including isoelectric focusing in IPG strips, with application of fewer than 2000 volt- hours, even as few as 1500 volt-hours.
- the methods may be capable of effecting electrophoretic separation, including isoelectric focusing in IPG strips, in fewer than 6 hours, 5 hours, even 4 hours or less.
- the potential difference may be applied 5 in a plurality of ramped voltage steps, in a plurality of stepped voltage steps, or at a constant voltage level.
- the strip may usefully be an IPG strip.
- the invention provides improved methods of electrophoresis using prior-cast, hydratable, separation media strips, wherein the improvement comprises spacedly o contacting the strips with an anode and cathode with resistance between power supply and gel sufficiently low as to permit electrophoretic separation with a maximum applied voltage of no more than 3000 volts, no more than 1500 volts, even no more than 500 volts.
- the strip is an immobilized pH gradient (IPG) strip, and the resistance is sufficiently low as to permit 5 isoelectric focusing with application of fewer than 2000 nominal volt-hours, even as few as
- the invention provides a buffer core device for forcibly urging an anode and a cathode into simultaneous spaced electrical communication with a plurality of prior-cast hydratable separation media strips enclosed within means that permit spaced o electrical communication separately with each of the enclosed strips through respective first and second entries.
- the device comprises a substantially inflexible frame; an anode support; a cathode support; an anode; and a cathode.
- the anode support and the cathode support are spacedly fixed to the frame and are capable of distributing an external compressive force respectively to the cathode and the anode to urge the cathode and the anode toward the enclosed strips with 5 greater contact pressure at the first and second entries than elsewhere on the enclosing means.
- the anode support may make intermittent contact with the anode and the cathode support may make intermittent contact with the cathode.
- FIG. 1 A is a front perspective view of one embodiment of a cassette of the present 5 invention
- FIG. 1 B is a front perspective view of another embodiment of a cassette of the present invention.
- FIG. 1 C is a front perspective view of the embodiment of FIG. 1 B, rendered as opaque; o
- FIG. 2 is a front perspective view of a cassette of the present invention with an IPG strip inserted into one of six available channels;
- FIG. 3A is a front perspective view of a cassette of the present invention, with well- forming member removed, prior to application of a conductive wick;
- FIG. 3B is a front perspective view of a cassette of the present invention with a first 5 conductive wick contacting the anodic end of IPG strips present in three of six available channels and a second conductive wick contacting the cathodic end of the three IPG strips;
- FIG. 3C is a back perspective view of an embodiment of a cassette of the present invention, particularly showing a recessed region that facilitates heat dissipation during electrophoresis; o [0064] FIG.
- FIG. 3D is a back perspective view of another embodiment of a cassette of the present invention, particularly showing a plurality of recessed regions that facilitate heat dissipation during electrophoresis;
- FIG. 4 is an exploded side perspective view of a multilaminate cassette of the present invention.
- FIG. 5 is an exploded side perspective view of a loading well assembly of a cassette of the present invention
- 5 is a front perspective view of a buffer core of the present invention, without anode electric wire or cathode electric wire, with gasket;
- FIG. 6B is a front perspective view of a buffer core of the present invention (front) operationally aligned to contact its anode and cathode electrodes respectively to anodic and cathodic wicks of a cassette of the present invention (rear); o [0069] FIG. 6C is a front perspective view of the buffer core and cassette of FIG. 6B in operational contact with one another;
- FIG. 6D is a front perspective view of a buffer core in operational contact with two cassettes of the present invention.
- FIG. 6E shows a buffer core of the present invention, with cassettes of the present 5 invention operationally engaged thereupon, further engaged in an electrophoresis chamber;
- FIG. 7A is a front view of a cassette of the present invention in which channel entries open through opposite surfaces of the cassette;
- FIG. 7B is a side view of the cassette of FIG. 7A;
- FIG. 7C is an exploded perspective view of two cassettes as shown in FIGS. 7A and 0 7B showing their operational relationship to a prior art buffer core;
- FIG. 7D is a perspective view of the cassettes of FIGS. 7A and 7B in operational contact with a prior art buffer core;
- FIG. 8 shows IPG strips after electrophoresis in channels of the stated internal dimensions; 5 [0077] FIG. 9 plots measured currents through IPG strips run, for comparison, in the cassette of the present invention using the buffer core of the present invention, and in a commercially available horizontal flat bed apparatus, using identical power supplies with identical voltage programs;
- FIG. 10 plots currents measured through IPG strips, and voltages reported by two o power supplies, using the cassette and buffer core of the present invention, with focusing performed using a stepped voltage profile; and [0079] FIG. 11 illustrates an electrophoresis tank cover useful for applying voltage to the cassette and buffer core of the present invention.
- the present invention is based, in part, upon the discovery that the swelling that attends rehydration of prior-cast hydratable separation media can be exploited to help lodge such media in an enclosure that permits spaced electrical communication with the enclosed separation medium.
- the spaced electrical communication makes it possible to apply a voltage gradient to the prior-cast hydratable separation media while the medium is lodged within the o enclosing member.
- the separation medium's contact with air is substantially reduced.
- the reduction in air contact obviates the prior art requirement for occlusive contact with a fluid oil layer during immobilized pH gradient isoelectric focusing.
- the prior-cast separation medium can be electrophoresed in any physical orientation.
- the prior-cast hydratable separation medium is an IPG strip
- relaxation of the prior-art requirement for horizontal electrophoresis makes it newly possible to perform IPG electrophoresis using the widely distributed, small footprint, vertical electrophoresis gel boxes presently used to perform SDS- 0 PAGE.
- the invention provides a method for performing electrophoresis, particularly for performing electrophoresis using prior-cast, hydratable separation media.
- electrophoresis explicitly includes isoelectric focusing. 5
- the method comprises hydratingly lodging a prior-cast hydratable electrophoretic separation medium within an enclosing member that permits spaced electrical communication with the enclosed media.
- the spaced electrical communication is used to apply a voltage gradient to the enclosed medium sufficient to effect electrophoretic separation of analytes therein.
- prior-cast electrophoretic separation medium refers to an electrophoretic separation medium, typically a polymeric gel, that has first been solidified, or gelled, elsewhere than in the enclosing member in which electrophoresis is to be performed.
- Electrophoretic separation media and methods of preparing, casting, and performing electrophoresis using electrophoretic media, are well known in the analytical arts, and need not be detailed here. See, e.g., Rabilloud (ed.), Proteome Research: Two-Dimensional Gel Electrophoresis and Identification Methods, Springer Verlag, 2000 (ISBN: 3540657924); Westermeier, Electrophoresis in Practice, 2nd ed,, John Wiley & Sons, 2000 (ISBN 3527300708); B.D. Hames et al.
- the "prior-cast electrophoretic separation medium" used in the present invention must have sufficient structural resiliency to be transferred or released from its casting mold and thereafter lodged within the enclosure of the present invention.
- such structural resiliency will be imparted to the separation medium by the adherence thereto or incorporation therein of a layer or lamina of another material, such as plastic.
- layers are known in the art, and include, e.g., polyester film backings, as are found in commercial IPG strips, and polyester mesh fabric, which can be incorporated into the separation medium.
- the "prior-cast electrophoretic separation medium” used in the present invention is typically fashioned as a strip — that is, with a first dimension substantially greater than a second dimension — such dimensions are not required for practice of the present invention. Nonetheless, for ease of description, all prior-cast electrophoretic separation media useful in the practice of the present invention are referred to in the alternative herein as "strips”.
- a "prior-cast hydratable electrophoretic separation medium” is a prior-cast electrophoretic medium that can be dehydrated and that, after rehydration, has retained sufficient structural integrity to permit electrophoretic separation of analytes there within.
- the prior-cast, hydratable, electrophoretic separation medium swell detectably after contact in its dehydrated state with an aqueous solution ("aqueous buffer", "buffer").
- aqueous buffer "buffer”
- the prior-cast hydratable electrophoretic separation medium will swell at least about 5% in volume, often at least about 10%, 15%, 20%, even at least about 25%, 30%, o 40% or more in volume upon contact with an aqueous buffer.
- the volume increase can be manifest in all three dimensions or, when the separation medium is backed with an inextensible layer, principally in one or in two dimensions.
- the volume increase can occur over a period of minutes or, in the case of IPG strips, more typically over a period of hours.
- the degree of swelling is sufficient if the prior-cast, hydratable, electrophoretic 5 separation medium swells sufficiently upon contact with an aqueous solution ("aqueous buffer", “buffer") as to permit hydratable lodging in an enclosing member.
- aqueous buffer aqueous buffer
- hydratable lodging is intended that the prior-cast, hydratable separation medium be insertable into an enclosing member in its dehydrated state, and that it become lodged in the enclosing member in its rehydrated state.
- the strip must be "insertable” in its dehydrated state, the strip need not necessarily be removable from the enclosing member in its dehydrated state.
- the rehydrated prior-cast hydratable separation medium is said to be "lodged" in the enclosing member (equivalently, "lodgingly enclosed” therein) when two conditions are met. First, the strip remains within the enclosing member when the enclosing member is brought into 5 vertical orientation. Second, when the enclosing member is brought into vertical orientation, at least 50% of the separation medium is precluded from direct communication with ambient atmosphere.
- frictional and surface tension forces between the rehydrated separation medium and the enclosing member can contribute to the strip's lodging therein, it is not intended that such frictional or surface tension forces be sufficient in o themselves to effect lodging of the strip within the enclosing member.
- the enclosing member will be sufficiently form-retaining as to be able to maintain dimensional integrity when maintained in contact with a prior-cast, hydratable separation medium that is swelling.
- the enclosing member is a cassette having a form-retaining channel cavity within which the prior-cast, hydratable separation medium is engaged.
- the enclosing member further permits spaced electrical communication with the 5 enclosed prior-cast hydratable separation medium. Communication can be direct, as by through-passage of anode and cathode electrodes, or indirect, as by passage of current through an intermediate polymer layer or wick, as will be further discussed below. [0100] After the prior-cast hydratable electrophoretic separation medium is lodged in the enclosing member, the spaced electrical communication is used to apply a voltage gradient o sufficient to effect electrophoretic separation of analytes therein.
- the prior-cast hydratable electrophoretic separation medium is typically inserted by the user in its dehydrated state in the enclosing member.
- the prior-cast hydratable separation medium such as an IPG strip
- the prior-cast hydratable separation medium is movably inserted by hand into a channel cavity present within the enclosing member.
- the prior-cast hydratable separation medium such as an IPG strip
- the prior-cast hydratable separation medium is movably inserted by hand into a depression, with the channel cavity o thereafter completed by closing the member.
- the dehydrated strip can be earlier-inserted during manufacture of the enclosing member, obviating insertion of the dehydrated prior-cast separation medium into the enclosing member by the user.
- the dehydrated separation medium is then contacted with an aqueous solution.
- the composition of the rehydration solution will depend upon the composition of the 5 sample and separation medium and the intended electrophoretic procedure, and its choice will thus depend on factors that are well known in the electrophoretic arts.
- the rehydration solution can usefully include urea, non-ionic or zwitterionic detergents, dithiothreitol o (DTT), dye, and a carrier ampholyte mixture suited to the pH range of the IPG strip.
- Carrier ampholyte mixtures for use in such rehydration solutions are available commercially (e.g., IPG Buffer pH 3.5 - 5.0, cat. no. 17-6002-02; IPG Buffer pH 4.5 - 5.5, cat. no.
- IPG Buffer pH 5.0 - 6.0 cat. no. 17-6002-05; IPG Buffer pH 5.5 - 6.7, cat. no. 17-6002-06; IPG Buffer pH 4 - 7, cat. no. 17-6002-86; IPG Buffer pH 6 - 11, cat. no. 17-6002-78; IPG Buffer pH 3 5 - 10 NL, cat. no. 17-6002-88; IPG Buffer pH 3 - 10, cat. no. 17-6002-87, all from Amersham Biosciences, Piscataway, NJ, USA),
- the rehydration solution can also advantageously include the sample intended to be separated in the prior-cast hydratable separation medium.
- the sample to be separated can be a mixture of proteins, such as those from a biological sample, and can usefully be or have been denatured, as by chaotropes, reducing agents, and detergents.
- the separation medium is other than an immobilized pH gradient strip
- the sample can include other types of macromolecules, such as nucleic acids.
- the methods of the present invention can include the later step of removing the prior- cast hydratable separation medium from the enclosing member after electrophoresis.
- the separation medium in certain embodiments of the methods of the present invention can be further analyzed within the enclosing member, o such as by staining and drying.
- the methods of the present invention include the use of an enclosing member that has (i) means for hydratingly lodging a prior-cast electrophoretic separation medium therewithin and (ii) means for spaced electrical communication with the enclosed separation medium, wherein the spaced electrical communication means can be used to apply a voltage gradient to the enclosed separation medium sufficient to effect electrophoretic separation of analytes present therewithin. 5 [0113] It is, therefore, another aspect of the present invention to provide an enclosing member useful in the practice of the methods of the present invention, which enclosing member is hereinafter called a "cassette".
- FIGS. 1A - 1C are front perspective views of embodiments of a cassette of the present invention.
- Cassette 100 comprises form-retaining member 10 and at least one channel 12 (in the embodiments shown in FIGS, 1A and 1B, cassette 100 has six substantially parallel channels 12, although fewer or greater numbers can be present), Form-retaining member 10 imparts dimensional integrity to prior-formed channels 12. Channels 12, although present, are not visible in FIG. 1C, rendered as fully opaque. 5 [0116] Referring again to FIG. 1A, channel 12 has first channel entry 14 and second channel entry 16 and cavity 18 therebetween.
- Cavity 18 of channel 12 is so dimensioned as to movingly engage a prior-cast hydratable electrophoresis medium ("strip"), such as an IPG strip, in its dehydrated state, and to lodgingly enclose the strip after hydration thereof.
- Strip hydratable electrophoresis medium
- First channel entry 14 and second channel entry 16 permit electrical communication o with cavity 18, and thus define a channel current flow axis through cavity 18.
- the channel current flow axis is in a plane substantially parallel to a substantially planar first surface of form-retaining member 10.
- rehydratable 5 electrophoresis strip 20 is inserted in its dehydrated state into channel
- strip 20 has been prior- inserted into cassette 100, either by the user or by the manufacturer thereof.
- Strip 20 is rehydrated within channel 12 by application of a rehydration solution, optionally containing the sample to be fractionated. o [0120] Rehydration solution is typically dispensed into channel 12 prior to insertion of strip
- Strip 20 since insertion of strip 20 into channel 12 is facilitated by wetting of the interior of channel 12.
- Strip 20 can, however, be prior-inserted into channel 12, with rehydration solution thereafter applied at either or both of entries 14 and 16.
- entry 14, entry 16, or both can be sealed — e.g. with tape or cover slip — to prevent evaporation and the accidental discharge of rehydration solution, [0121]
- strip 20 Upon rehydration, strip 20 becomes lodged in cavity 18 of channel 12, at least in part 5 due to swelling of the separation medium. Strip 20 is thereafter not readily removed from channel 12 without expansion of cavity 18, as further described below.
- sample is then applied at entry 14, entry 16, or both with the cassette oriented horizontally to retain sample, and allowed to enter the separation medium.
- o sample can be prior-absorbed into a wick which is then inserted into entry 14, entry 16, or both, from which wick the sample then enters the separation medium, As further described below, sample entry can be facilitated by application of electrical current.
- Electrophoresis is then performed by applying a voltage gradient to strip 20, causing current to flow along the channel current flow axis.
- strip 20 is typically removed from channel 12 for further processing, such as staining and/or contacting of strip 20 (or a portion thereof) to a gel to effect separation along a second dimension.
- Removal is typically effected by expansion of cavity 18 using a method appropriate to the composition of cassette 100; for example, in embodiments of cassette 100 in which one or more laminae contribute to the circumferential walls of cavity 18, removal can be o effected by peeling of the laminae, thus opening channel 12.
- further processing can be effected within channel 12.
- form-retaining member 10 is constructed of form-retaining nonliquid materials. Preferred materials are those that are readily machined, molded, or etched, that are chemically compatible — that is, do not suffer substantial degradation upon 5 contact — with electrophoretic buffer systems, that do not appreciably bind or impede the transport of analytes through the enclosed gel, and that provide a vapor gas barrier.
- form-retaining member 10 can be constructed from translucent, or transparent material, including optical quality transparent material, thus permitting strip 20 to be visualized while engaged in cavity 18.
- form-retaining member 10 is constructed of materials that are o substantially electrically nonconducting, thus reducing or eliminating the concurrent action on strip 20 of electrical fields other than those along the channel current flow axis through cavity [0126]
- form-retaining member 10 is composed of ceramic, quartz, glass, silicon and its derivatives, plastic, or mixtures thereof.
- plastics useful in the construction of form-retaining member 10 are polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethylmethacrylate, polyvinylchloride, polytetrafluoroethylene, polystyrene, 5 polycarbonate, polyacetal, polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose, polystyrene, polyacrylonitrile, polyurethane, polyamides, polyaniline, polyester, and mixtures or copolymers thereof,
- Form-retaining member 10 is also usefully composed of materials that permit heat to be conducted away from strip 20 during electrophoresis.
- form-retaining member 0 10 can usefully be shaped to include one or more recessed regions 27, shown in FIGS. 3C and 3D, reducing the thickness of form-retaining member 10 in regions proximal to channels 12, reducing thermal resistance between strip 20 and a heat sink, usefully a fluid filled chamber, as further discussed below.
- Form-retaining member 10 confers dimensional integrity upon channels 12.
- Form-retaining member 10 can confer dimensional integrity upon channel 12 by contributing at least a portion of the circumferential wall of cavity 18 of channel 12.
- cavity 18 of channel 12 can be constructed as a tunnel, bore, or conduit within form-retaining member 10, In such embodiments, form-retaining member 10 contributes the entirety of the circumferential wall of cavity 18.
- cavity 18 can be partially enclosed within form-retaining member 10, with only a portion of the circumferential cavity wall of cavity 18 contributed by member 10.
- channels 12 can be machined into form-retaining member 10, or, depending on the composition of form-retaining member 10, lithographed, engraved, isotropically or anisotropically etched, milled, mechanically or chemically polished, or molded into form-retaining member 10.
- channels 12 can be fabricated on form-retaining member 10 from silicon or resin deposits or slabs.
- FIG. 4 is an exploded side perspective view of a multilaminate embodiment of cassette 100 of the present invention.
- form-retaining member 10 includes depression 13
- Laminate cover 42 includes a plurality of entries 50.
- depression 13 becomes fluidly enclosing along cavity 18, thus completing channel 12, with entries 50 contributing to channel entries 14 and 16.
- laminate cover 42 can usefully be optically translucent or transparent, and is usefully substantially electrically insulating.
- laminate cover 42 can be composed of ceramic, i o quartz, glass, silicon and its derivatives, alumina, polymer, plastic, or mixtures thereof.
- plastics useful in the construction of laminate cover 42 are polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethylmethacrylate, polyvinylchloride, polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal, polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose, polystyrene, polyacrylonitrile, polyurethane, polyamides, polyaniline, polyester,
- Laminate cover 42 can usefully be, and is often preferably, flexible. Although laminate cover 42 can be of any thickness, to confer flexibility laminate cover 42 can usefully be a film. [0138] Laminate cover 42 can be attached to form-retaining member 10 by bonding means known in the microfabrication arts, including thermal welding, ultrasonic welding, and o application of adhesives or adhesive layers.
- U.S. Patent Nos. 5,800,690 and 5,699,157 describe methods for completing channels by attaching planar cover elements to micromachined substrates by thermal bonding, application of adhesives, or by natural adhesion between the two components.
- U.S. Patent No. 5,593,838, incorporated 5 herein by reference teaches that localized application of electric fields permits the meltable attachment of a cover element at about 700°C, well below the flow temperature of silicon (about 1400°C) or of Corning 7059 glass (about 844°C).
- WO 96/04547 (Lockheed Martin Energy Systems), incorporated herein by reference in its entirety, teaches that a cover plate can be bonded directly to a glass substrate after treatment in dilute NH4OH/H2O2, followed by 0 annealing at 500°C, well below the flow temperature of silicon-based substrates.
- WO 98/45693
- Laminate cover 42 is usefully attached to form-retaining member 10 by reversible 5 bonding means, thus permitting the user to separate laminate cover 42 from form-retaining member 10 after completion of electrophoresis, which in turn permits strip 20 to be removed from channel 12 for further processing. Constructing laminate cover 42 as a flexible film offers advantages in such user-mediated separation of laminate cover 42 from form-retaining member 10. o [0141] In the embodiment depicted in FIG. 4, laminate cover 42 is attached adhesively to form-retaining member 10 using double-sided laminate adhesive layer 46. [0142] As shown, double-sided laminate adhesive layer 46 has elongate slots 48 that are congruent with depressions 13. Such slots 48 prevent contact between double-sided adhesive layer 46 and strip 20 when strip 20 is movably inserted into channel 12; contact with adhesive 5 can interfere with movable insertion of strip 20 into cassette 100,
- laminate cover 42 is itself fashioned as a form-retaining member, typically thicker than the flexible film above-described. In some of these embodiments, laminate cover 42 is fashioned as a discrete structure.
- form-retaining laminate cover 42 and form-retaining member 10 are movably attached to one other, as by a hinge, or plurality of hinges, present therebetween,
- the hinge 5 need not itself be fashioned as a separate, intermediating, structure, but can instead be fashioned as a foldable seam between form-retaining member 10 and laminate cover 42. Such seams are common in plastic cases designed to hold, e.g., drill bits.
- laminate cover 42 In cases in which laminate cover 42 is form-retaining, it can be assembled to form- retaining member 10 by, e.g., snapping laminate cover 42 to form-retaining member 10.
- a o pressure compliant surface, on form-retaining member 10 and/or laminate cover 42 facilitates sealing of the two layers, forming an enclosing member suitable for electrophoresis, Although assembly by snapping of laminate cover 42 to form-retaining member 10 has been described with particularity, any other mechanical engagement approach, such as mating of tongue and groove, insertion of a tab into a slot, etc., can also be used to similar effect.
- any other mechanical engagement approach such as mating of tongue and groove, insertion of a tab into a slot, etc., can also be used to similar effect.
- the internal diameter of cavities 18 can be adjusted by 5 adjusting the depth of incursion of channel 12 into form-retaining member 10.
- Channel 12 is so dimensioned — in both multilaminate and unitary embodiments of o cassette 100 — as to permit insertion of a prior-cast hydratable strip-based electrophoresis medium, such as an IPG strip, in its dehydrated state, and to lodgingly enclose the strip after hydration.
- a prior-cast hydratable strip-based electrophoresis medium such as an IPG strip
- channel 12 of cassette 100 will have a width of at least about 3.0 mm, 3.1 mm, 3,2 mm, 3.3 mm, 3.4 mm, and even 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3,9 mm, 4.0 mm, and even 4.1 mm, and will have depth of 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 even 0.7 mm, 0.71 mm, 0.72 mm, 0.73 mm, 0.74 mm, o 0.75 mm, 0.76 mm, and even
- ReadyStrip IPG strips presently available commercially from Bio-Rad (Hercules, CA, USA) have strip width of 3.3 mm and gel thickness of 0.5 mm. Accordingly, to permit electrophoresis of these commercial IPG strips, channel 12 of cassette 100 will have an 5 approximate width of at least about 3.3 mm, 3.4 mm, and even and even 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm, and even 4.1 mm, and will have depth of 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 even 0.7 mm, so as to movingly engage such strips in their dehydrated state and lodgingly enclose the strips when rehydrated.
- channels 12 have width of 3.7 mm and depth of 0.64 mm, [0151] As would be expected, prior-cast hydratable electrophoretic separation media can, and likely will, be manufactured with dimensions different from those presently used.
- cassettes 100 of the present invention are not limited to those dimensioned for use with the above-described strips.
- channel 12 Dimensions of channel 12 are suitable if, in step (2), strip 20 can be advanced into channel 12 to a point at which less than 1 cm of strip 20 remains outside the entry chosen for insertion, and if, in step (3), air does not directly contact more than 50% of the enclosed separation medium. Strip 20 should also remain lodged within the cassette once the cassette is brought vertical in step (3).
- the swelling of the separation medium causes direct, occlusive, contact of the separation medium with the channel's internal wall along substantially all of the channel cavity.
- a visibly labeled solution such as 0.2% w/v bromphenol blue in water
- a visibly labeled solution will typically not extend more than about 0.25 cm beyond the channel entry into the channel cavity.
- the swelling of the separation medium is insufficient to cause occlusive contact of the separation medium with the channel's internal wall along substantially all of the channel cavity,
- a visibly labeled solution such as 0.2% w/v bromphenol blue in water will enter the channel cavity from the superior entry when the cassette is brought vertical. In neither case, however, will air contact more than 50% of the enclosed separation medium.
- An additional, functional test for suitability of the internal dimensions of channel 12 for a prior-cast hydratable electrophoretic separation medium of given dimensions is to replace step (3) of the test set forth above with an actual electrophoresis experiment; dimensions of channel 12 are suitable if, in step (2), strip 20 can be advanced into channel 12 to a point at 5 which less than 1 cm of strip 20 remains outside the entry chosen for insertion, and if, after electrophoresis, adequate electrophoretic separation is achieved.
- strip 20 is an IPG strip, this latter test may usefully be performed as follows. [0161] Mix 5.0 ⁇ L of Serva IEF standard (catalogue no.
- [0163] Contact the cassette to the electrodes of a buffer core (further described hereinbelow). Apply a buffer dam (further described below) to the other contact face of the buffer core. Slide o the buffer core into an electrophoresis chamber and fill the outer chamber surrounding the buffer core with water. Take care that the water does not overtop the cassette and spill into the inner chamber (the outer walls of which are defined by the cassettes and buffer core). [0164] Apply a voltage in three steps according to the following profile: 200 V for 20 min, 450 V for 15 minutes, 750 V for 15 minutes, 2000 volts for 30 minutes. 5 [0165] Channel dimensions are suitable if discrete marker bands are observable.
- Channel entries 14 and 16 will typically, but not invariably, be spaced so that channel 12 engages substantially the entire length of strip 20, as shown e.g. in FIG. 2.
- IPG strips are currently available commercially in a variety of lengths. For example, Immobiline DryStrip IPG strips, presently commercially available from Amersham Biosciences, o (Piscataway, NJ, USA), are available with gel lengths of 70 mm, 110 mm, 130 mm, 180 mm, and 240 mm. ReadyStrip IPG strips, presently commercially available from Bio-Rad (Hercules, CA, USA), are available with gel lengths of 70 mm, 110 and 170 mm. ZOOM® IPG strips presently commercially available from Invitrogen (Carlsbad, CA, USA) have gel lengths of 70 mm.
- channels 12 are fashioned to accommodate substantially the entire length of strips with gel lengths of 70 mm, 110 mm, 170 mm, 180 mm, and 240 mm in length.
- channels 12 will typically have length at least as long as the stated gel length (70, 100, 170, 180, or 240 mm), typically with extension of 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, or even 6 mm on both ends.
- channel 12 will be at least about 70 mm in length, 72 mm in length, 74 mm in length, 76 mm in length, 78 mm in length, and even 80 or 82 mm in length.
- channel 12 will be 80 mm in length.
- the overextending portion(s) of strip 20 will be exposed to atmospheric CO2, obviating an important advantage of the present invention.
- the overextending portion(s) of strip 20 can permit leakage of ampholyte and/or protein from the strip. Additionally, only that portion of the separation medium lying between the spaced electrical connections will be functionally available for separation, reducing the functional portion of gel. Finally, the overextending portion(s) might interfere mechanically with establishment of electrical communication properly required for electrophoresis. And when strip 20 underextends channel 12, it can prove difficult to establish effective electrical communication with the enclosed strip.
- strip 20 overextends channel 12 excess can be removed using scissors or knife; typically, only that portion of strip 20 lacking separation media will be so removed. If strip 20 underextends channel 12, the recessed end can be brought into effective electrical communication with the exterior of channel 12 by filling the recessed end with an electrically conductive, channel-filling, material,
- the material can be a polymer gel, such as agarose.
- the agarose can be rendered molten in the presence of electrolyte-containing buffer, such as o rehydration solution, applied to entry 14, entry 16, or both as a molten liquid, and thereafter allowed spontaneously to gel with decrease in temperature.
- electrolyte-containing buffer such as o rehydration solution
- Polyacrylamide can also be used, although in this latter case polymerization of monomers and cross-linkers must be effected by addition of catalyst, as is well known in the art.
- cassette 100 includes a plurality of channels 12.
- the current flow axes of plural channels 12 are usefully substantially parallel to one another, and cavities 18 of plural channels 12 are fluidly noncommunicating with one another except at channel entries 14 and 16.
- channels 12 need not have identical cavity 18 dimensions, a single cassette 100 thus accommodating strips 20 of different dimensions. Typically, however, o cavities 18 of plural channels 12 will all have the same internal dimensions.
- cassette 100 is described above as permitting user-directed insertion of strip 20 into channel 12, it is another aspect of the present invention to provide a cassette, as above-described, in which strips 20 have already been inserted during manufacture. Such cassettes 100 can usefully be disposable. 5 [0179] To facilitate sample application, and in particular to facilitate sample application without cross contamination as among plural channels 12, cassette 100 can usefully include loading wells.
- FIG. 1A shows one embodiment of such loading wells;
- FIGS. 1B and IC show another embodiment of such loading wells.
- cassette 100 is shown to have two well-forming members o 22. The two well-forming members define discrete reservoirs, termed loading wells, at each of the six entries 14 and six entries 16, respectively. When cassette 100 is horizontal with well- forming members 22 superior to form-retaining member 10, each loading well can maintain a defined maximum volume of fluid in contact with an entry 14 (or entry 16) without cross-over fluid contact with adjacent entries,
- the loading wells permit samples of volume less than the maximum reservoir volume to be applied 5 discretely to individual wells 14 (and/or 16) without cross-over contamination. In cases in which sample is applied in rehydration buffer prior to insertion of strips 20 into channels 12, the loading wells prevent cross-over contamination by sample displaced from channel 12 during strip insertion.
- sample to be fractionated such as a protein sample for isoelectric focusing on o IPG strips
- well-forming members 22 can be removable. Such removal can facilitate subsequent application of conductive wicks 24, as shown in FIG. 3B and further 5 described below.
- well-forming member 22 is typically removed prior to electrophoresis, there are fewer constraints on the materials from which it can be constructed than for form-retaining member 10 and, in multilaminate embodiments of cassette 100, for laminate cover 42. Indeed, well-forming member 22 can be constructed of any material that is substantially chemically o unreactive with the rehydration solution, such as ceramic, quartz, glass, silicon and its derivatives, plastic, natural or synthetic rubber polymers, or mixtures thereof.
- well-forming member 22 plastics useful in the construction of well-forming member 22 are polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethylmethacrylate, polyvinylchloride, polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal, polysulfone, celluloseacetate, cellulosenitrate, 5 nitrocellulose, polystyrene, polyacrylonitrile, polyurethane, polyamides, polyaniline, and mixtures thereof. Silicone and its derivatives are also useful. [0184] In certain embodiments, well-forming member 22 can be composed of electrically conductive materials; this facilitates "active rehydration" of strip 20. In “active rehydration", strip 20 is rehydrated in the presence of a low voltage gradient, approximately 100 V, o established along the channel current flow axis of strip 20 between entries 14 and 16.
- well-forming member 22 can be composed of an electrically-conductive material, such as an electrically-conductive polymer, such as a polymer impregnated or doped with carbon.
- an electrically-conductive material such as an electrically-conductive polymer, such as a polymer impregnated or doped with carbon.
- cassette 100 is unitary — that is, having channels 12 formed completely within form-retaining member 10 — well-forming members 22 can be attached to form-retaining member 10.
- cassette 100 is, instead, multilaminate — e.g., with channels 12 formed in o part by a laminate cover 42 — well-forming members 22 can be attached to laminate cover 42, as shown in FIG. 5.
- FIG. 5 is an exploded side perspective view showing well-forming members 22 attached adhesively to laminate cover 42 using double-sided well-forming member adhesive layer 54.
- well-forming member 22 can be attached to laminate cover 42 by a variety of bonding means well known in the microfabrication arts, including thermal welding, ultrasonic welding, and application of liquid or partially cured adhesives, as well as by means of adhesive layers.
- Well-forming member 22 can in the alternative be attached to laminate cover 42 by o engagement of opposing, matching surfaces, as in a snap, or engagement of tongue with groove, or engagement of tab with slot.
- well forming members 22 will usefully be reversibly attached to cassette 100, thus permitting removal of the well-forming members prior to electrophoresis.
- adhesive layer 5 54 the adhesive layer is usefully designed to adhere more strongly to form-retaining member
- FIG. 3A shows an embodiment of the cassette of the present invention with well- forming members 22 removed, prior to application of conductive wicks, as further described below.
- sealing is accomplished by application to entries 14 and/or 16 of a material that is electrically conductive, that can be applied in a state in which it conforms in shape to the entry and/or loading well, and that thereafter polymerizes or gels into a shape-holding phase.
- a material that is electrically conductive that can be applied in a state in which it conforms in shape to the entry and/or loading well, and that thereafter polymerizes or gels into a shape-holding phase.
- such material can usefully be a polymer gel, such as agarose or acrylamide.
- cassette 100 can optionally, and usefully, include ribs 40. 5 [0195] Ribs 40 facilitate alignment of laminate cover 42 and well-forming members 22 during manufacture of cassette 100.
- Ribs 40 can also facilitate proper operational engagement of cassette 100 by an electrophoresis chamber or buffer core, as further described below, [0196] Ribs 40 can be machined or molded directly from form-retaining member 10, or can be separately constructed and fixed thereto. When separately constructed, ribs 40 are usefully o constructed of solid or semisolid materials that are readily machined, molded, or etched, and that are chemically compatible — that is, do not suffer substantial degradation upon contact — with electrophoretic buffer systems. Usefully, ribs 40 can be constructed of materials that are substantially electrically insulating, including ceramic, quartz, glass, silicon and its derivatives, or plastic, or mixtures thereof.
- plastics useful in the construction of ribs 40 are 5 polymethylacrylic, polyethylene, polypropylene, polyacrylate, polymethylmethacrylate, polyvinylchloride, polytetrafluoroethylene, polystyrene, polycarbonate, polyacetal, polysulfone, celluloseacetate, cellulosenitrate, nitrocellulose, polystyrene, polyacrylonitrile, polyurethane, polyamides, polyaniline, polyester, and mixtures and copolymers thereof.
- strip 20 o becomes lodgingly enclosed in cavity 18 of channel 12.
- FIG. 3B illustrates one useful, but nonlimiting, approach by which cassette-enclosed strip 20 is rendered contactable by cathode and anode electrodes to complete the necessary 5 electrical circuit.
- FIG. 3B is a front perspective view of a cassette of the present invention having six channels 12. As shown, a first conductive wick 24 contacts strips 20 (present in three of six available channels 12) at entries 14; a second conductive wick 24 contacts strips 20 at entries 16. o [0200] Wick 24 includes an electrically conductive material. The material need not be constitutively conductive: it suffices, and indeed typically will be the case, that wick 24 is conductive when wet. In this latter case, wick 24 can usefully be composed of a bibulous material, such as paper, nitrocellulose, felt, nylon, or derivatives thereof.
- strip 5 20 can be electrically coupled to cathode and anode electrodes through intermediation of electrically conductive polymers or hydrogels such as agarose.
- first conductive wick 24 can usefully contact each of plural entries 14, and second conductive wick 24 can usefully contact each of plural entries 16, facilitating application of current in parallel to plural channels 12. While useful, such geometry 0 is not required.
- First conductive wick 24 is then contacted with an electrode, serving as either cathode or anode.
- an electrode serving as either cathode or anode.
- the choice as between applying a cathode or anode to wick 24 depends upon the intended electrophoretic technique, the location of sample application, and other conditions well known to those in the electrophoretic arts. For example, for isoelectric focusing using IPG 5 strips, where one end of the strip is acidic and the other basic, the basic end of the strip is preferably placed in electrical communication with the cathodic electrode.
- Second conductive wick 24 is then contacted with an electrode (an anode if first wick 24 is contacted with the cathode, a cathode if first wick 24 is contacted with the anode).
- Any means of electrode attachment to wicks 24 can be used, as long as effective o electrical communication is established.
- spaced electrical communication with enclosed strip 20 can be effected by direct contact of strip 20 with anode and cathode electrodes. Contact can be accomplished by passage of anode and cathode electrodes through entries 14 or 16, or alternatively by passage of electrodes through form-retaining member 10 or laminate cover 42 elsewhere than at entries 14 and/or 16. As an example of the latter approach, electrodes shaped as blades can be used to pierce laminate cover 42 in 5 embodiments in which laminate cover 42 is a flexible film, thereby contacting enclosed strip 20 at spaced intervals.
- Electrophoresis can thereafter be conducted with cassette 100 in any physical orientation.
- electrode contact is effected using an adaptor that permits electrophoresis to be conducted with cassette 100 maintained vertically; even o when cassette 100 is held vertical, channels 12 of cassette 100 can be horizontal or vertical, as desired.
- modular electrophoresis systems for performing slab gel electrophoresis in the vertical dimension are well known, see e.g. U.S. Patent Nos. 5,888,369 and 6,001 ,233, and are commercially available (Invitrogen, Carlsbad, CA, USA; Bio-Rad, Hercules, CA, USA).
- the adaptor of the present invention permits cassettes 100 of the present invention to be electrophoresed in such existing modular electrophoresis systems, permitting the efficient use of such prior-purchased equipment for electrophoresis of prior-cast hydratable electrophoretic separation media, such as IPG strips.
- FIG. 6A is a front perspective view of an adaptor, herein termed a buffer core, of the o present invention.
- FIG. 6B is a front perspective view of the buffer core (front) operationally aligned with, but not yet contacting, a cassette of the present invention (rear); operational contact is shown in FIG. 6C,
- buffer core 26 is designed simultaneously to align cathode electrode wire 31 with cathodic wick 24 of cassette 100 and anode electrode wire 32 with anodic wick 24 of cassette 100.
- cathode wire 31 is attached at a first end to cathode contact prong 38; analogously, anode wire 32 is attached at a first end to anode contact prong 36.
- Contact prongs 38 and 36 permit the removable attachment of wires having standard female gender plugs; as is well known in the electrophoresis arts, the other end of such wires is typically connected to a power supply, such as a regulatable power supply.
- cathode wire 31 extends from cathode contact prong 38 to support 28 before terminating at a second end, and anode wire 32 extends from anode contact prong 36 to o support 30 before terminating at a second end.
- Supports 28 and 30 are typically composed of materials that are substantially electrically insulating and substantially inert to electrophoresis running buffers. For example, supports 28 and 30 are conveniently made of plastic, such as polycarbonate.
- buffer core 26 can, and typically will, be operationally aligned and contacted simultaneously with a second cassette 100. So aligned and so contacted, buffer o core 26 and cassettes 100 define an internal chamber 62, open only at the top and sealed, except from above, from external liquids.
- cassettes 100 are aligned and contacted to buffer core 26, The assembly is then engaged in electrophoresis buffer chamber 34 which itself, or in conjunction with an additional device, urges cassettes 100 (or singular cassette 100 and buffer dam) into sealable contact with buffer core 26.
- additional urging device can be a cam-activated clamp ("tension wedge"), as further described in U.S. Pat. No.
- buffer core 26 is first loosely engaged in electrophoresis buffer chamber 34, and cassettes thereafter aligned, contacted to, and then further urged against buffer core 26.
- buffer core 26 and cassettes 100 are typically, but optionally, further facilitated by a gasket, such as a silicone gasket, fitted into groove 70 of buffer core 26, shown in FIGS. 6A and 6D.
- a gasket such as a silicone gasket
- buffer core 26 and cassettes 100 in sealed engagement therewith define internal chamber 62. This chamber isolates cathode wire 31 and anode wire 32 from fluids present external to buffer core 26 in i o electrophoresis chamber 34 (chamber 60), so long as the fluid level in electrophoresis chamber 60 does not over top cassettes 100.
- electrophoresis chamber 34 can be filled with any chosen liquid solution, to a level that does not overtop cassettes 100, without affecting the electrical circuit, Such fluids can thus usefully serve as a heat sink, reducing the temperature of strips 20 as they are
- Electrophoresis is conducted by attaching, via contact prongs 36 and 38, anode and cathode to a power supply. Conveniently, this may be performed by applying a cover having integrated electrodes, as illustrated in FIG. 11.
- the cover may usefully be designed so as to fit existing electrophoresis chambers, permitting their use with the present invention, but to o interface only with buffer cores of the present invention.
- a potential difference is applied that is sufficient to effect separation of analytes within the separation medium of strip 20.
- proteins influenced by the voltage gradient, begin to migrate until the pi of the protein coincides with the pH on the immobilized gradient, at which point the focused protein 5 ceases to move.
- parasitic impedances include, e.g., (i) cable connections to the power o supply, (ii) resistances in the cables themselves, (iii) cable-to-electrode contact resistance,
- the invention provides apparatus that provides a resistance pathway between power supply and prior-cast hydratable separation medium that is 5 substantially lower than that found in prior art apparatus used for electrophoresis in the prior art; the reduced resistance substantially increases the efficiency with which prior-cast hydratable separation media, such as IPG strips, may be electrophoresed.
- the electrode-to-wick and wick-to-gel contact resistances can contribute significantly to the overall voltage drop from power supply to gel. These contact o resistances depend upon the respective contact pressures. At any given compressive force applied inwardly against cassettes 100, the magnitude of these electrically effective contact pressures will depend upon the proportion of the force brought to bear at these locations. Accordingly, supports 28 and 30 may usefully be designed to distribute the external compressive force discontinuously, creating greater contact pressure at the first and second 5 channel entries than elsewhere on cassette 100.
- the cassette contact faces of support 28 and support 30 are nonplanar: a plurality of discontinuous indentations collectively define a series of intervening protuberances.
- the serrated surface so created is capable of discontinuously distributing external compressive force to anode wire 31 o and cathode wire 32.
- the indentations are sufficiently deep as to cause periodic discontinuities in the contact of the electrode wires to their respective supports. Such depth of indentation is not required.
- Indentations are positioned so as to align each intervening protuberance with an entry 5 14 (equivalently for the other electrode, entry 16) of cassette 100. Indentations are sized so as to create protuberances with dimensions closely approximated to the lateral dimensions of entries 14 and 16.
- supports 28 and 30 have (on at least one cassette-contact face) six protuberances, each positioned to align with one of the six entries 14 (equivalently, entries 16) of cassette 100, and preferably sized to as to approximate the lateral dimensions of entries 14 and 16.
- support 28 and support 30 that contact the same cassette 100 typically will have the same number of protuberances. However, for each of supports 28 and 30, the 5 two cassette contact faces need not have the same number of protuberances (as is the case in the embodiments illustrated in FIGS. 6A - 6C), if two cassettes 100 accommodating different numbers of strips 20 are to be applied to the two cassette contact faces. Typically, however, the two cassette contact faces will have the same number of protuberances.
- support 28 and support 30 lack o indentations. Instead, the supports are nonunitary in construction, comprising materials that are differentially compressible.
- the least compressible materials are positioned to align with entries 14 (equivalently 16) of cassette 100; the more compressible materials are positioned to align elsewhere on cassette 100.
- the different degrees of compressibility cause differential distribution of the external compressive force, causing increased contact pressure at entries 14 5 (and 16).
- the discontinuously applied pressures - occasioned, for example, by serrating the outward surfaces of supports 28 and 30, as in FIGS. 6A - 6C - substantially improve the efficiency with which voltage (and current) can be applied to strips 20.
- Efficiency of voltage (and current) application to IPG strips defined herein as the ratio, for a given voltage output o from a power supply, of currents measured at identical strips 20 having identical samples, may be at least 2-fold better, 3-fold better, 4-fold better, even at least 5-fold better using the cassette and buffer core of the present invention as compared to horizontal, oil immersion IPG electrophoresis devices of the prior art.
- Efficiencies may even be at least 6-fold, 7-fold, 8-fold, 9-fold, and even at least 10-fold better.
- the data set forth in Examples 2 and 3 herein, plotted 5 in FIGS. 9 and 10, demonstrate a 2 - 4 fold better efficiency than is observed using a prior art oil immersion flatbed IPG device.
- the increased efficiency of electrical transmission provides significant advantages.
- the increased efficiency permits IPG IEF to be performed using substantially lower power supply voltages and power supply currents, permitting less expensive power supplies, o lacking current limitation means, to be used.
- the data set forth in Example 3 below demonstrates that even simple unregulated power supplies capable only of step voltage profiles may be used, obviating the need for power supplies capable of ramped voltage profiles.
- the increased efficiency permits shorter run times to achieve the volt-hours required for focusing.
- Focusing can be achieved in as few as 6 hours, 5 hours, 4 hours, even as few as 3.5 hours, 3.0 hours, 2.5 hours, 2.0 5 hours, or even as few as 1.5 or 1 ,25 hours, depending upon the sample, strip length, strip pH range, and voltage profile. Focusing can thus typically be achieved in 1.25 - 10 hours, 1.5 - 9 hours, 1.75 - 8 hours, 2 - 7 hours, 2.5 - 6 hours, and even in 1 - 3 hours. [0237] Focusing can thus be achieved at least two times faster than with existing horizontal flatbed oil-immersion devices, often at least 3-times, 4-times, even 5-times faster. At times, o focusing can be achieved at least 6-times, 7-times, 8-times, 9-times, even as much as 10-times faster.
- focusing can be achieved using the following ramped voltage profile: 5 [0239] 0 - 175 volts over 15 minutes [0240] 175 - 2000 volts over 45 minutes [0241] 2000 volts for 20 - 30 minutes,
- IPG o isoelectric focusing can be achieved using maximal voltages as low as 3500 V, 3000 V, 2750
- Minimal voltages may be 500 V, 750 V, 1000 V, 1250 V, 1500 V, 1750 V, 2000 V, 2250 V, even 2500 V or more.
- the increased efficiency of the cassette and buffer core of the present invention permit focusing to be achieved in fewer than 13,000 nominal volt-hours, typically in fewer than 12,000 nominal volt-hours, 11 ,000 volt hours, 10,000 nominal volt hours, 9000 nominal volt hours, 8000 nominal volt hours, 7000 nominal volt-hours, 6000 nominal volt-hours, 5000 5 nominal volt-hours, 4000 nominal volt-hours, 3000 nominal volt-hours, even as few as 2000, 1900, 1800, 1700, 1600, 1500, 1400 or as few as 1300, 1200, 1100, or 1000 nominal volt- hours, for strips of 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 for shorter, longer
- buffer core 26 with cassettes 100 can be stored in a sealed container at -80°C until strips are ready for analysis.
- Strips 20 can, and typically will, be withdrawn from cavity 18 for further processing, with or without prior freezing. As described earlier, although strips 20 can at times be removed o upon drying via channel entries 14 and 16, strips 20 will typically be removed by expanding the dimensions of cavity 18 of channel 12; in multilaminate embodiments of cassette 100, this is accomplished by separating laminate cover 42 from form-retaining member 10.
- the buffer core embodiment above-described is designed to facilitate electrophoresis of a cassette in which, for each channel present therein, channel entries 14 and 16 permit 5 electrical communication with the channel cavity 18 therebetween through a common surface of cassette 100, as is shown, e.g., in FIGS. 1 - 3.
- FIG. 7A is a front view, and FIG. 7B a side view, of a cassette 1000 of the present invention in which entries 114 and 116 of channels 112 respectively open through opposite surfaces of cassette 1000.
- Channels 112 of cassette 1000 are so dimensioned as to movingly engage a prior-cast hydratable electrophoretic separation medium in its dehydrated state and lodgingly enclose the strip after rehydration.
- cathode and anode must establish electrical communication with strip 20 o from opposite sides of cassette 1000.
- entries 14 and 16 open channel 12 to the same face of cassette 100, so too electrical communication of channel 112 through entries 114 and 116 can be direct, as by through-passage of electrodes through respective entries, or indirect, as by intermediation by polymer gels and/or conductive wicks. Additionally, however, when entries 114 and 116 open on opposite sides of cassette 1000, electrical communication can be established by contact of anode and cathode electrodes separately to a first and a second buffer reservoir, which 5 reservoirs in turn separately contact entries 114 and 116.
- first and second buffer reservoirs must be maintained in electrical isolation from one another, except by way of a circuit to be completed through the separation medium of strip 20, [0266]
- Such geometry can readily be effected by sealingly contacting cassettes 1000, or a o singular cassette 1000 and a buffer dam, to a buffer core 126, as further described in commonly-owned U.S. Patent No. 5,888,369, incorporated herein by reference in its entirety, and as available commercially from Invitrogen Corp. (XCell IITM Buffer Core with Electrodes, catalogue no. EI9014X, Invitrogen Corp., Carlsbad, CA),
- two cassettes 1000 are lodgingly engaged in operational alignment with buffer core 126, as shown in FIGS, 7C and 7D.
- buffer core 126 is first loosely engaged in electrophoresis buffer chamber 34, and cassettes thereafter aligned, contacted to, and then further urged against buffer core 126.
- Fluid-tight contact between buffer core 126 and cassette 1000 (or a buffer dam) is 5 typically, but optionally, further facilitated by a gasket, such as a silicone gasket, fitted into groove 170 of buffer core 126.
- Buffer core 126 and cassettes 1000 in sealed engagement therewith define internal chamber 162 which, if cassettes 1000 are not overtopped, is fluidly noncommunicating with electrophoresis buffer chamber 34.
- a conductive o solution is then added to internal chamber 162 to a level that (i) contacts cassette entries 114
- a conductive solution is also added to electrophoresis buffer chamber 34 to a level that (i) contacts the cassette entries 116 (or 114, as the case may be) that open into chamber 34, and (ii) that does not overtop cassettes 1000.
- the electrode geometry of buffer core 126 effects 5 contact of the anode to internal chamber 126 and cathode to an external reservoir 60 formed in chamber 34, thus permitting the requisite voltage gradient to be applied across strip 20 to effect electrophoresis.
- cassettes of the present invention have been particularly described herein above as having at least one prior-formed channel with sufficient dimensional integrity as to permit the lodging by hydration of prior-cast hydratable separation media engaged there within, prior-formed channels are only one approach to hydratingly lodging such media within 5 an enclosing member.
- the enclosing member if malleable yet shape-retaining, can be wrapped around the strip in its dehydrated form, fashioning a de novo channel which, upon hydration of the strip, lodgingly encloses the rehydrated strip there within.
- the present invention provides kits that facilitate the practice of the o methods of the present invention.
- kits of the present invention may consist of at least one component (singularly or as a plurality thereof) selected from the group consisting of: a buffer core of the present invention, an electrophoresis chamber, an electrophoresis chamber lid that can establish electrical communication with a buffer core, a tension wedge, a buffer dam, an electrode wick, 5 sealing tape, an IPG strip, containerized carrier ampholytes, containerized cathode buffer, containerized anode buffer, containerized stains (such as silver stains or colloidal blue stains), analyte standards, such as protein standards, typically in admixture, and a polyacrylamide gel suitable for performing a second dimension of separation.
- a buffer core of the present invention an electrophoresis chamber
- an electrophoresis chamber lid that can establish electrical communication with a buffer core, a tension wedge, a buffer dam, an electrode wick, 5 sealing tape, an IPG strip, containerized carrier ampholytes, containerized cathode buffer, containerized anode buffer,
- a first series of kit embodiments may include nondisposable items useful o for adapting electrophoresis devices to accommodate cassettes of the present invention.
- a kit may include a buffer core of the present invention and corresponding lid.
- a kit of the present invention may include an electrophoresis chamber, tension wedge, buffer core and lid.
- the kit may 5 additionally optionally comprise a buffer dam.
- a second series of kit embodiments may include disposable items suitable for use in the invention.
- a kit may include at least one cassette and a plurality of electrode wicks.
- the kit may optionally additionally comprise sealing tape suitable for sealing entries 14 and 16 o during rehydration, and/or may optionally contain a plurality of IPG strips, either with identical pH ranges or differing pH ranges.
- Kits of the present invention may also, optionally, include any one or more of separately containerized carrier ampholytes, cathode buffer, anode buffer, stains (such as silver stains or colloidal blue stains) - either in liquid form, at 1X use concentration or higher 5 concentration for further dilution, or in dry form to be reconstituted with water of suitable quality - protein standards, typically in admixture, or polyacrylamide gels suitable for performing a second dimension of separation following isoelectric focusing, such as a Tris- Glycine ZOOM® gel (Invitrogen, Carlsbad, CA).
- stains such as silver stains or colloidal blue stains
- Three cassettes were manufactured by machining six parallel channels each into 5 form-retaining plastic slabs, with geometry essentially as shown in FIG. 1 A.
- the six channels of the first cassette all were 0.77 mm in depth, with two channels 4.09 mm in width, two channels 0.65 mm in width, and two channels 3.35 mm in width.
- the six channels of the second cassette all were 0.65 mm in depth, with two channels 4,09 mm in width, two channels 0.65 mm in width, and two channels 3.35 mm in width.
- the six channels of the third cassette o were 0.57 mm in depth, with two channels 4.09 mm in width, two channels 0.65 mm in width, and two channels 3.35 mm in width.
- the channels were rendered fluidly enclosing except at terminal entries by application of a flexible laminate cover to each of the three cassettes.
- Serva IEF standard 5 ⁇ L, (catalogue no. 39212-01 , Serva Electrophoresis GmbH, Heidelberg, Germany) was mixed with 120 ⁇ L of rehydration buffer of the following 5 composition: 8.0 M urea, 0.5% ampholytes (3-10 IPG buffer, cat. no. 17-6001-11 , Amersham Biosciences), 2.0% (w/v) CHAPS, 20 mM DTT, 0.0025% (w/v) bromphenol blue. The solution was pipetted into each channel of the three cassettes, with the cassette positioned horizontally.
- a filter paper wick dampened with water was placed in contact with the extreme ends of the gel portion of the strip at the terminal entries.
- Electrodes were contacted to the wick at the anodic and cathodic ends of the cassette 5 and a voltage applied in three steps according to the following protocol: 250 volts for 15 minutes, ramp from 250 - 3500 volts for 1 hour and 30 minutes, and 3500 volts for 1 hour, Current was limited to 1 mA and power to 4 watts in all three steps.
- Strips were removed, stained with Coomassie blue stain, aligned, and photographed.
- Results shown in FIG. 8 indicate that even the largest channel, 4.09 mm in width and o 0.77 mm in depth, permitted adequate focusing of the Serva IEF standard (left-most lane) in strips with nominal width of 3 mm and depth of 0,5 mm.
- DMMs were used in current-measurement mode. They were set up in series with the ground-side power cable between the apparatus and the power supply.
- Table 2 presents "volt-hour” ratios for the two types of devices during the constant
- Table 2 demonstrates that the cassette and buffer core of the present invention are far more efficient at transferring power to the gels than is the flatbed MultiphorTM, despite apparent identity in the nominal volt-hours calculated by multiplying power supply voltage by time.
- the cassette and buffer core of the present invention pushes up to 4 times the amount of current through the strips (both AP and INV strips) than the MultiphorTM does. There were visible differences in the velocity and shape of the dye fronts between the two apparatuses during this stage.
- the cassette and buffer core of the present invention delivered an average of approximately 2.6 times and 2.9 times more current to the AP and INV strips, respectively, than did the MultiphorTM.
- the cassette and buffer core of the present invention delivered an average of approximately 2.6 times and 2.9 times more current to the AP and INV strips, respectively, than did the MultiphorTM.
- lower voltages and times may be used to achieve focusing with the cassette and buffer core of the present invention than has been used for prior flat bed devices.
- Lower voltages for shorter periods are more convenient, require less sophisticated (and expensive) power supplies, obviate the need for current limiting power supplies, and should lead to less frequent arcing and thus burning of the strips.
- Optimal voltages and times will, as before, depend in part on sample osmolality and electrolyte concentration,
- the stepped voltage profile resulted in no burning of any of the 24 strips.
- the 500 volt step resulted in 99 ⁇ amps per strip for less than a minute, but was over 70 ⁇ amps for approximately 5 minutes; however, 500 volts is a significantly lower voltage than the final focusing voltage, and the conductivity of the sample at this low voltage is not likely to l o cause any arcing or burning, No arcing or burning was seen.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Electrochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Electrostatic Separation (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
Claims
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002489725A CA2489725A1 (en) | 2002-06-18 | 2003-06-17 | Methods and apparatus for low resistance electrophoresis of prior-cast, hydratable separation media |
EP03760464A EP1525464A2 (en) | 2002-06-18 | 2003-06-17 | Methods and apparatus for low resistance electrophoresis of prior-cast, hydratable separation media |
AU2003253661A AU2003253661A1 (en) | 2002-06-18 | 2003-06-17 | Methods and apparatus for low resistance electrophoresis of prior-cast, hydratable separation media |
JP2004513742A JP2005530151A (en) | 2002-06-18 | 2003-06-17 | Method and apparatus for low resistance electrophoresis of precast hydratable separation media |
US12/485,379 US8034223B2 (en) | 2001-05-10 | 2009-06-16 | Methods and apparatus for low resistance electrophoresis of prior-cast, hydratable separation media |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US39025902P | 2002-06-18 | 2002-06-18 | |
US60/390,259 | 2002-06-18 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2003106973A2 true WO2003106973A2 (en) | 2003-12-24 |
WO2003106973A3 WO2003106973A3 (en) | 2004-03-25 |
Family
ID=29736693
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2003/019335 WO2003106973A2 (en) | 2001-05-10 | 2003-06-17 | Methods and apparatus for low resistance electrophoresis of prior-cast, hydratable separation media |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP1525464A2 (en) |
JP (1) | JP2005530151A (en) |
CN (1) | CN1675538A (en) |
AU (1) | AU2003253661A1 (en) |
CA (1) | CA2489725A1 (en) |
WO (1) | WO2003106973A2 (en) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112354572A (en) * | 2019-07-11 | 2021-02-12 | 北京理工大学 | Multidimensional microfluidic electrophoresis chip, detection device and detection method |
US11156603B2 (en) | 2010-04-05 | 2021-10-26 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11162132B2 (en) | 2015-04-10 | 2021-11-02 | Spatial Transcriptomics Ab | Spatially distinguished, multiplex nucleic acid analysis of biological specimens |
WO2021236625A1 (en) * | 2020-05-19 | 2021-11-25 | 10X Genomics, Inc. | Electrophoresis cassettes and instrumentation |
US11208684B2 (en) | 2010-04-05 | 2021-12-28 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11286515B2 (en) | 2013-06-25 | 2022-03-29 | Prognosys Biosciences, Inc. | Methods and systems for determining spatial patterns of biological targets in a sample |
US11352659B2 (en) | 2011-04-13 | 2022-06-07 | Spatial Transcriptomics Ab | Methods of detecting analytes |
US11624086B2 (en) | 2020-05-22 | 2023-04-11 | 10X Genomics, Inc. | Simultaneous spatio-temporal measurement of gene expression and cellular activity |
US11733238B2 (en) | 2010-04-05 | 2023-08-22 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5906519B2 (en) * | 2011-09-13 | 2016-04-20 | 国立大学法人 熊本大学 | Protein separation method by two-dimensional electrophoresis |
US10578580B2 (en) * | 2016-09-23 | 2020-03-03 | University Of Notre Dame Du Lac | One-step capillary isoelectric focusing and mobilization of analytes |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6113766A (en) * | 1997-06-09 | 2000-09-05 | Hoefer Pharmacia Biotech, Inc. | Device for rehydration and electrophoresis of gel strips and method of using the same |
-
2003
- 2003-06-17 CN CN 03818732 patent/CN1675538A/en active Pending
- 2003-06-17 JP JP2004513742A patent/JP2005530151A/en not_active Withdrawn
- 2003-06-17 AU AU2003253661A patent/AU2003253661A1/en not_active Abandoned
- 2003-06-17 WO PCT/US2003/019335 patent/WO2003106973A2/en active Application Filing
- 2003-06-17 CA CA002489725A patent/CA2489725A1/en not_active Abandoned
- 2003-06-17 EP EP03760464A patent/EP1525464A2/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6113766A (en) * | 1997-06-09 | 2000-09-05 | Hoefer Pharmacia Biotech, Inc. | Device for rehydration and electrophoresis of gel strips and method of using the same |
Cited By (37)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11732292B2 (en) | 2010-04-05 | 2023-08-22 | Prognosys Biosciences, Inc. | Spatially encoded biological assays correlating target nucleic acid to tissue section location |
US11365442B2 (en) | 2010-04-05 | 2022-06-21 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11542543B2 (en) | 2010-04-05 | 2023-01-03 | Prognosys Biosciences, Inc. | System for analyzing targets of a tissue section |
US11519022B2 (en) | 2010-04-05 | 2022-12-06 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11208684B2 (en) | 2010-04-05 | 2021-12-28 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11733238B2 (en) | 2010-04-05 | 2023-08-22 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11293917B2 (en) | 2010-04-05 | 2022-04-05 | Prognosys Biosciences, Inc. | Systems for analyzing target biological molecules via sample imaging and delivery of probes to substrate wells |
US11549138B2 (en) | 2010-04-05 | 2023-01-10 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11313856B2 (en) | 2010-04-05 | 2022-04-26 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11560587B2 (en) | 2010-04-05 | 2023-01-24 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11156603B2 (en) | 2010-04-05 | 2021-10-26 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11634756B2 (en) | 2010-04-05 | 2023-04-25 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11371086B2 (en) | 2010-04-05 | 2022-06-28 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11384386B2 (en) | 2010-04-05 | 2022-07-12 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11767550B2 (en) | 2010-04-05 | 2023-09-26 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11401545B2 (en) | 2010-04-05 | 2022-08-02 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11866770B2 (en) | 2010-04-05 | 2024-01-09 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11479810B1 (en) | 2010-04-05 | 2022-10-25 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11761030B2 (en) | 2010-04-05 | 2023-09-19 | Prognosys Biosciences, Inc. | Spatially encoded biological assays |
US11479809B2 (en) | 2011-04-13 | 2022-10-25 | Spatial Transcriptomics Ab | Methods of detecting analytes |
US11352659B2 (en) | 2011-04-13 | 2022-06-07 | Spatial Transcriptomics Ab | Methods of detecting analytes |
US11788122B2 (en) | 2011-04-13 | 2023-10-17 | 10X Genomics Sweden Ab | Methods of detecting analytes |
US11795498B2 (en) | 2011-04-13 | 2023-10-24 | 10X Genomics Sweden Ab | Methods of detecting analytes |
US11359228B2 (en) | 2013-06-25 | 2022-06-14 | Prognosys Biosciences, Inc. | Methods and systems for determining spatial patterns of biological targets in a sample |
US11286515B2 (en) | 2013-06-25 | 2022-03-29 | Prognosys Biosciences, Inc. | Methods and systems for determining spatial patterns of biological targets in a sample |
US11618918B2 (en) | 2013-06-25 | 2023-04-04 | Prognosys Biosciences, Inc. | Methods and systems for determining spatial patterns of biological targets in a sample |
US11753674B2 (en) | 2013-06-25 | 2023-09-12 | Prognosys Biosciences, Inc. | Methods and systems for determining spatial patterns of biological targets in a sample |
US11821024B2 (en) | 2013-06-25 | 2023-11-21 | Prognosys Biosciences, Inc. | Methods and systems for determining spatial patterns of biological targets in a sample |
US11613773B2 (en) | 2015-04-10 | 2023-03-28 | Spatial Transcriptomics Ab | Spatially distinguished, multiplex nucleic acid analysis of biological specimens |
US11162132B2 (en) | 2015-04-10 | 2021-11-02 | Spatial Transcriptomics Ab | Spatially distinguished, multiplex nucleic acid analysis of biological specimens |
US11739372B2 (en) | 2015-04-10 | 2023-08-29 | Spatial Transcriptomics Ab | Spatially distinguished, multiplex nucleic acid analysis of biological specimens |
US11390912B2 (en) | 2015-04-10 | 2022-07-19 | Spatial Transcriptomics Ab | Spatially distinguished, multiplex nucleic acid analysis of biological specimens |
US11299774B2 (en) | 2015-04-10 | 2022-04-12 | Spatial Transcriptomics Ab | Spatially distinguished, multiplex nucleic acid analysis of biological specimens |
CN112354572A (en) * | 2019-07-11 | 2021-02-12 | 北京理工大学 | Multidimensional microfluidic electrophoresis chip, detection device and detection method |
WO2021236625A1 (en) * | 2020-05-19 | 2021-11-25 | 10X Genomics, Inc. | Electrophoresis cassettes and instrumentation |
US11624086B2 (en) | 2020-05-22 | 2023-04-11 | 10X Genomics, Inc. | Simultaneous spatio-temporal measurement of gene expression and cellular activity |
US11866767B2 (en) | 2020-05-22 | 2024-01-09 | 10X Genomics, Inc. | Simultaneous spatio-temporal measurement of gene expression and cellular activity |
Also Published As
Publication number | Publication date |
---|---|
CA2489725A1 (en) | 2003-12-24 |
AU2003253661A1 (en) | 2003-12-31 |
JP2005530151A (en) | 2005-10-06 |
EP1525464A2 (en) | 2005-04-27 |
CN1675538A (en) | 2005-09-28 |
AU2003253661A2 (en) | 2003-12-31 |
WO2003106973A3 (en) | 2004-03-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8034223B2 (en) | Methods and apparatus for low resistance electrophoresis of prior-cast, hydratable separation media | |
US6936150B2 (en) | Methods and apparatus for electrophoresis of prior-cast, hydratable separation media | |
US20210063346A1 (en) | Electro-blotting devices, systems, and kits and methods for their use | |
CN107209144B (en) | Dry protein transfer | |
EP0324539A2 (en) | Improved capillary gel electrophoresis columns | |
WO2003106973A2 (en) | Methods and apparatus for low resistance electrophoresis of prior-cast, hydratable separation media | |
WO2007106832A2 (en) | Multifunctional electrophoresis cassette | |
EP0979403A1 (en) | Membrane loader for gel electrophoresis | |
JP5236609B2 (en) | Sample separation adsorption device | |
US10101296B2 (en) | Mini-gel comb | |
AU2002245701A1 (en) | Methods and apparatus for electrophoresis of prior-cast, hydratable separation media | |
Görg et al. | Two-dimensional electrophoresis with immobilized pH gradients | |
Fujii et al. | A Simple and Easy-to-Use Capillary Isoelectric Focusing Technique Using Reagent-Release Hydrogels | |
JP2003114216A (en) | Electrophoretic device | |
WO2007106370A9 (en) | Transfer device for two - dimensional electrophoresis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2489725 Country of ref document: CA Ref document number: 2004513742 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2003760464 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2003253661 Country of ref document: AU |
|
WWE | Wipo information: entry into national phase |
Ref document number: 537705 Country of ref document: NZ |
|
WWE | Wipo information: entry into national phase |
Ref document number: 20038187329 Country of ref document: CN |
|
WWP | Wipo information: published in national office |
Ref document number: 2003760464 Country of ref document: EP |