WO2003058226A1

WO2003058226A1 - Methods and compositions for isoelectric focusing

Info

Publication number: WO2003058226A1
Application number: PCT/US2003/000438
Authority: WO
Inventors: Michel Assenheimer; Haim Moskowitz
Original assignee: Proteologics, Inc.
Priority date: 2002-01-07
Filing date: 2003-01-07
Publication date: 2003-07-17
Also published as: AU2003202918A1

Abstract

In certain aspects, the invention provides improved methods and apparatus for isoelectric focusing. Such methods may be used in combination with other methods to perform, for example, two-dimensional electrophoresis. In many embodiments, the invention provides methods and apparatus that permit isoelectric focusing in the absence of oil-based or hydrophobic cover layers. Methods and apparatus of the invention provide for more rapid isoelectric focusing.

Description

METHODS AND COMPOSITIONS FOR ISOELECTRIC FOCUSING

Background

Isoelectric focusing (IEF) is a powerful tool for the study of biomolecules such as proteins. The process of IEF separates proteins on the basis of isoelectric point, or the pH at which a protein carries a net charge of zero. While IEF may be used as a stand-alone technique for protein analysis or in combination with a variety of analytical methods, it is most commonly used as the first resolving step, or dimension, in two-dimensional electrophoresis (2-D electrophoresis). In 2D- electrophoresis, proteins are first resolved by isoelectric focusing, followed by an orthogonal SDS-PAGE electrophoresis that separates proteins by size. The combination of these two resolving steps provides a powerful system for the separation and subsequent identification of hundreds to thousands of proteins.

2D-electrophoresis has become a pivotal analytical tool in the growing field of proteomics, primarily because it permits the rapid separation of large numbers of proteins from a single sample. Several technological advancements have made 2D- electrophoresis a simpler, more reproducible, and hence more popular method. Such advancements include the replacement of carrier ampholyte-based gradients with immobilized pH gradients (IPGs), and the commercial availability of highly uniform IPG strips or gels.

Despite these advancements, IEF remains a time consuming process. In addition, the separation of hydrophobic proteins, such as membrane proteins or lipoproteins, remains problematic. With the increased use of 2D-electrophoresis in proteomics research, there is a great need for more rapid and reliable IEF systems.

It is an object of the invention to provide methods and apparatus for improved IEF. In particular, the invention provides methods and apparatus for improved IEF of samples containing lipophilic proteins. Summary

In certain aspects, the invention provides apparatus and methods for conducting isoelectric focusing, electrophoresis and rehydration using a gel strip. In preferred embodiments, an apparatus of the invention is designed such that isoelectric focusing, electrophoresis and rehydration can be performed without the use of a hydrophobic cover fluid to cover the gel strip.

In certain embodiments, the invention provides an apparatus comprising: (a) a tray having a first surface and at least one channel formed in the first surface, the channel having a length, width, depth and a floor and being sized and shaped to receive a hydrated gel strip having a first and second surface, wherein the length and width of the channel is substantially equal to the length and width of the gel strip, and; (b) a cover having a first surface and at least one first electrode compartment and at least one second electrode compartment formed in the first surface, the at least one first electrode compartment and the at least one second electrode compartment being spaced apart on the first surface of the cover a distance substantially equal to the length of the channel, wherein the first surface of the cover is sized and shaped as to mate the first surface of the tray substantially sealing the channel formed in the first surface of the tray to form an elongated enclosure, and wherein the electrodes contact the surface of the gel strip directly or by a mediating filter, and (c) a mechanical assembly for receiving the tray and the cover comprising a means for mating the first surface of the tray to the first surface of the cover, and whereby the first surface of the cover can be disjoined and rejoined in a sealing relationship with the first surface of the tray, wherein when the gel strip is positioned on the floor of the channel and the first surface of the cover is mating the channel in a sealing relationship, the entire first surface of the gel strip is in close contact with the floor of the channel and the entire second surface of the gel strip is in close contact with the first surface of the cover, except for those portions of the first surface of the cover containing the first and second electrode compartments.

In certain aspects, the invention provides an apparatus comprising: (a) tray having a first surface and at least one channel formed in the first surface, the channel having a length, width, depth and a floor and being sized and shaped to receive a rehydrated gel strip having a first and a second surface, wherein the length and width of the channel is substantially equal to the length and width of the gel strip, and wherein at least one first electrode compartment and at least one second electrode compartment are formed in the floor of each of the at least one channel, the at least one first electrode compartment and the at least one second electrode compartment being spaced apart on the floor of the at least one channel a distance substantially equal to the length of the channel; and (b) a cover having a first surface sized and shaped as to mate the first surface of the tray substantially sealing the channel formed in the first surface of the tray to form an elongated enclosure and (c) a mechanical assembly for receiving the tray and the cover comprising a means for mating the first surface of the tray to the first surface of the cover and whereby the first surface of the cover can be disjoined and rejoined in a sealing relationship with the first surface of the tray, wherein when the gel strip is positioned on the floor of the channel and the first surface of the cover is mating the channel in a sealing relationship, the entire first surface of the gel strip is in close contact with the floor of the channel, except for those portions of the floor of the channel containing the first and second electrodes, and the entire second surface of the gel strip is in close contact with the first surface of the cover.

In certain aspects, the invention provides an apparatus comprising: (a) a tray having a first surface and at least one channel formed in the first surface, the channel having a length, width, depth and a floor and being sized and shaped to receive a gel strip and rehydration buffer, and wherein the length and width of the channel is substantially equal to the length and width of the gel strip, and (b) a cover for the tray having a first surface sized and shaped as to mate the first surface of the tray sealing the channel formed in the first surface of the tray to form an elongated enclosure, and (c) a mechanical assembly for receiving the tray and the cover comprising a means for mating the first surface of the tray to the first surface of the cover, and whereby the first surface of the cover can be disjoined and rejoined in a sealing relationship with the first surface of the tray, wherein when the gel strip is positioned on the floor of the channel and the first surface of the cover is mating the channel in a sealing relationship, and when the gel strip is rehydrated, the entire first surface of the gel strip is in close contact with the floor of the channel and the entire second surface of the gel strip is in close contact with the first surface of the cover.

In certain preferred embodiments of the above disclosed apparatus, the distance between the floor of the channel and the first surface of the cover is less than the thickness of the gel strip when rehydrated (without mechanical constraint or without oil cover or any other hydrophobic cover fluid) and wherein the distance between the floor of the channel and the first surface of the cover permits the rehydration of the gel strip to at least 80% of its thickness when rehydrated (without mechanical constraint or without oil cover or any other hydrophobic cover fluid).

The channel of any of the above disclosed apparatus may have a cross- section of any shape, although a quadrilateral cross-section, such as a rectangle or trapezoid is illustrated in the Drawings herein. In certain embodiments, at least one end of the channel is tapered, and optionally the cathode is applied at the tapered end.

In certain aspects, the application provides a method for isoelectric focusing separation comprising: (a) providing a gel strip, wherein the gel strip has a length, width, height, and a first surface and a second surface both defined by the length and width of the gel strip; (b) placing said gel strip on the floor of a channel formed in a tray; (c) providing a solid detachable cover for the channel, wherein the cover and/or the floor of the channel comprises a first and second electrode compartment; (d) covering the gel strip with said cover, wherein when the gel strip is positioned on the floor of the channel and the cover is mating the tray in a sealing relationship, the entire first surface of the gel strip or the plastic support is touching the floor of the channel and the entire second surface of the gel strip is touching the cover, except at the first and second electrode compartments, wherein the electrodes contact the surface of the gel strip without the plastic support directly or by a mediating filter, (e) loading a sample onto the gel strip before, after or concurrently with any of the parts of the method and (f) applying an electrical field across the gel strip so as to obtain an isoelectrically focused sample. Optionally, the method may further comprise an initial rehydration process such as a rehydration process comprising: (a) providing a dry gel strip, (b) placing said gel strip on the floor of a channel formed in a tray, (c) bathing said gel strip with a rehydration buffer, (d) providing a solid detachable cover for the channel and (e) covering the gel strip and rehydration buffer with said cover in a sealing relationship, wherein when the gel strip is rehydrated, the entire first surface of the gel strip is touching the floor of the channel and the entire second surface of the gel strip is touching the cover, except for any electrode compartments, if applicable. Optionally, at the completion of the rehydration process the gel is rinsed. Optionally, the detachable cover can be disjoined and rejoined in a sealing relationship with the tray. Optionally, the cover for the tray in contact with the gel is coated. Optionally, the coating is a silanization or a hydrophilic coating. Optionally, crystallites remaining after rehydration are rinsed away.

In certain aspects the methods disclosed herein are used to resolve proteins in a sample, wherein the sample comprises, in small or large part, proteins of a category selected from the group consisting of: membrane proteins, hydrophobic proteins, protein having one or more lipophilic regions when denatured, proteins difficult to solubilize in aqueous environments, proteins requiring solubilized by high concentrations of chaotropes for their solubilization and, proteins solubilized with detergents. Optionally, a rehydration buffer contains chaotropes in concentrations sufficient to solubilize proteins that are otherwise difficult to solubilize. Optionally, a rehydration buffer contains urea and thiourea.

In certain embodiments, a gel strip is selected from the group consisting of: a gel strip containing an immobilized pH gradient and a gel strip containing two or more ampholytes, although other types of gel strips are also contemplated for use in one or more embodiments of the invention. In certain embodiments, the gel strip comprises a support that forms the first or second surface of the gel strip.

Brief Description Of The Drawings

These and other features and advantages of the invention will be more fully understood by reference to the following detailed description in conjunction with the attached drawings in which like reference numerals refer to like elements through the different views. The drawings illustrate principles of the invention and, although not to scale, show relative dimensions.

Figure 1 is a perspective view of an apparatus for conducting isoelectric focusing according to the present invention, illustrating the cover removed from the tray of the apparatus;

Figure 2 is a front view in cross section of the apparatus of Figure 1, illustrating the cover positioned on the tray of the apparatus;

Figure 3 is an elevational view of the bottom surface of the cover of the apparatus of Figure 1, illustrating the electrode compartments formed in the cover;

Figure 4 is side view in cross section of the apparatus of Figure 1;

Figure 5 is a side view in cross section of the tray of the apparatus of Figure 1, illustrating a plate covering the tray;

Figure 6 is an elevational view of the top surface of a multi-channel tray of an alternative embodiment of an apparatus for conducting isoelectric focusing according to the present invention;

Figures 7A and 7B are elevational views of the bottom surface of alternative covers for use with the multi-channel tray of Figure 6;

Figure 8 represents a perspective view of an apparatus for conducting isoelectric focusing according to another embodiment of the present invention, illustrating the cover removed from the tray of the apparatus;

Figure 9 is a perspective view of another embodiment of the apparatus, illustrating an alternative embodiment of the cover and the tray of the apparatus;

Figures 10A and 1 OB are a exemplary embodiments of the apparatus.

Detailed Description Of Certain Embodiments Overview:

In certain aspects, the invention relates to methods and apparatus for IEF, rehydration and/or electrophoresis. In various embodiments, the invention provides methods for performing any of the various steps of IEF in the absence of a cover layer of hydrophobic fluid such as an oil-based fluid or kerosene. This includes steps of rehydration of a gel strip as well as steps of focusing of proteins in a gel strip containing an immobilized pH gradient (IPG), carrier ampholytes or other IEF or electrophoresis methods. The term "gel strip" is used herein to refer to any gel of dimensions and composition suitable for protein analytic techniques such as IEF or electrophoresis. A "gel strip" may be dehydrated or hydrated. A "gel strip" may include one or more supports, such as a plastic support, positioned along one or more sides of the gel strip. Where a gel strip is said to be in contact with another element, such as the floor of a chamber, it may be the support portion of the gel strip that is actually in contact with that element. Exemplary gel strips include IPG gel strips and gel strips containing carrier ampholytes. A gel strip will generally have a length that is substantially greater than the width or depth. A gel strip will generally have a flattened rectangular cross-section, but may also have a cylindrical or other cross-section that is compatible with protein analytic techniques.

In further aspects, the invention provides methods for performing any of the steps of IEF with a buffer that is conductive, and often aqueous. In addition, the invention provides apparatus for use with IEF procedures generally, and particularly suited to use with the methods described herein.

Advantages of the methods and apparatus described herein are numerous, hi certain aspects, the methods and apparatus described herein permit the detection and separation of substantially more proteins as measured by the number of spots. In certain further aspects, the methods described herein permit substantially decreased runtime for successful IEF, as measured by volt-hours. In some embodiments, runtime is decreased by at least 25%, at least 35%, at least 40%, at least 50%, and least 60%, at least 70% at least 80%, or at least 100%. In addition, the methods and apparatus provided herein, in certain embodiments, give better resolution of the separated proteins, which may be visualized as tighter, more discrete spots and/or bands. Specifically, an advantage of the method and apparatus, as applied to IEF, relates to the substantially decreased spot size and hence increased resolution and separation capacity. Even with significantly reduced runtime the separation resolution is substantially improved, hi yet other aspects, the invention provides IEF methods for improved separation of hydrophobic proteins, such as, for example, lipoproteins and/or proteins having membrane spanning domains. In yet another aspect of the invention the method and device permit the separation and detection of membrane or membrane associated proteins and detergent-solubilized proteins. Moreover, another aspect of the invention provides a method and device for more rapid isoelectric focusing. A further advantage of this invention is a method and device providing for uniform and controlled rehydration of gel strips. In yet other aspects, the invention provides for a method and device for performing the rehydration of dry gel strips or the casting of gel strips, followed by a subsequent isoelectric focusing step of the IEF procedure. A further advantage of the invention is a method and device allowing efficient rinsing between the two steps of the isoelectric focusing separation procedure: the rehydration step of the dry gel strip and the focusing step. In addition, thanks to the electrode paper, the proposed apparatus enables narrow range focusing without any effect of proteins reaching the electrodes, as well as wide range focusing with IPG or Ampholynes. h a further embodiment of the invention, the method and device tapers out the isoelectric focusing medium's end conditions, which is advantageous e.g. for minimizing cathodic drift.

While not wishing to be bound by theory, it seems probable that the conventional use of a hydrophobic cover layer in contact with the gel strip causes hydrophobic proteins to partition towards the more hydrophobic regions at the interface between the gel strip and the hydrophobic cover layer. Thus, proteins with hydrophobic domains might be washed away during further processing of the gel strip. Such events may interfere with efficient, high-resolution separation of such proteins and may also result in loss of these proteins from the IEF separation. Moreover, while not wishing to be bound by theory, we suggest that detergents and/or other amphiphilic molecules may in part partition to the hydrophobic phase. Consequently, the effective detergent concentration in the sample as well as the matrix may change. Thus, sample components, such as proteins, may be in less favorable conditions and may precipitate and hence not be separated. Electrophoresis causes the proteins to migrate while the hydrophobic force holds them in place. The combination of the two effects results in a slower migration of the proteins during the focusing process.

While not wishing to be bound by theory, hydrophilic - hydrophobic interfaces are clearly detrimental to the IEF process, in particular for amphiphilic membrane proteins and detergents. Therefore, in certain aspects, the invention provides a system of IEF that avoids the use of oil-based fluids that may be difficult to clean up and may require special waste disposal systems, hi addition, a hydrophobic cover layer may interfere with the loading or reswelling of the sample onto the gel strip. Accordingly, in certain aspects the invention provides methods and apparatus for ease of loading samples.

IEF Methods

In certain aspects, the invention provides methods for performing any of the different steps of IEF or a combination thereof.

Generally, IEF procedures are performed with a gel strip supporting a pH gradient.

In certain aspects, IEF procedures are performed with an immobilized pH gradient. In certain embodiments, such gradients are created by using bifunctional compounds. The bifunctional compounds have a first function of being able to polymerize or otherwise form stable associations with the matrix through which the proteins or other materials will be electrophoresed. The bifunctional compounds have a second function of providing a weakly acidic or basic moiety. By creating a gradient of the bifunctional compounds and stably associating the gradient with the matrix, it is possible to generate an immobilized pH gradient. Exemplary bifunctional compounds are the acrylamido buffers, wherein the first functional portion of the molecule is an acrylamide subunit that can polymerize with a polyacrylamide gel matrix, and wherein the second functional portion is a weak acid (often a carboxylate group) or a weak base (often an amido group). Such acrylamido buffers are commercially available through Amersham Pharmacia as hnmobiline™ reagents.

Generally the gel strip will be polymeric and typically a gel, such as polyacrylamide. The matrix may be formed into any desired shape, although it is generally preferable to have a shape that is substantially longer in one dimension than in the other two dimensions. For example, the gel strip may be shaped like a long, thin cylinder or it may be shaped like a thin, narrow strip. Optionally, the gel strip may be affixed to a different material for improved stability. For example, the gel strip may be affixed to a support or semi-enclosing material such as plastic. Ready-made IPG gel strips with a plastic support are available from Amersham Biosciences under the name hnmobiline™ DryStrip gels. An IPG gel strip may be prepared with either a broad pH gradient such as 3-12 or a narrow pH gradient such as 5-6, 9-12, or 10-12. Exemplary pH gradients may also range from pH 4-7, 6-11, 3-10, etc. Such gradients may be linear or non-linear, depending on the type of application desired. In view of this specification, one of skill in the art will appreciate any suitable alternatives to the matrices and compounds disclosed herein.

In certain aspects, IEF procedures can be performed with carrier ampholytes (such as: Ampholine, Pharmalyte, Servalyte etc.). In certain embodiments, pH gradients are created by using low molecular weight bifunctional molecules and two electrode buffers. The bifunctional compounds consist of polyamino and polycarboxylic acids having an excellent buffer capacity. Those molecules are introduced to the gel either when the gel is cast or rehydrated. A pre-ran step is performed. In this step a pH gradient is created between acidic and basic environments of the electrode-buffers at each end of the gel. By applying a voltage to the electrodes positioned at the extremities of the gel, the small ampholyte molecules migrate to and focus at their isoelectric point. Once those molecules stop migrating they create a local pH environment, creating a pH gradient across the gel. Samples for use in IEF may be prepared in any of a number of ways. Often, samples will contain protein, but may contain many compounds in addition to protein, and may be free of protein. Protein-containing samples may be denatured or non-denatured, although denatured proteins typically provide superior separation, hi general, if protein samples are to be used, it is desirable to have a preparatory step, such as, for example, an ammonium sulphate precipitation, a trichloroacetic acid precipitation, an acetone precipitation, etc. When it is desirable to separate and detect a diverse profile of proteins, it is preferable to select preparatory steps that minimize the selective removal of certain proteins over others. Denaturation, if desired, may be achieved through the use of chaotropes and/or non-ionic or zwitterionic detergents. Appropriate chaotropes include urea and thiourea, or a mixture thereof. Appropriate detergents include NP-40, Triton X-100, CHAPS, etc. It may also be desirable to include a reductant, such as dithiothreitol (DTT) or beta- mercaptoethanol (BME) or Tris(carboxyethyl)phosphine (TCEP). Other variations in sample preparation will, in view of this specification, be apparent to one of skill in the art.

In certain aspects, an IPG gel strip will be provided as a pre-made gel strip, and optionally the pre-made gel strip will be dried for easy storage and/or shipment. For example, Immobiline™ DryStrips (Amersham Pharmacia Biotech) are provided as a dry polyacrylamide gel strip. Dry matrices will typically require a rehydration step prior to electrophoresis. hi certain embodiments, sample loading (see below) may be done concunently with the rehydration step.

In certain embodiments, rehydration is achieved by placing a gel strip in a tray having at least one suitably sized receptacle portion, such as a channel formed in the tray. An exemplary tray is a rehydration/electrophoresis tray of the invention, as described in detail below in connection with Figures 1-5 and Figures 8-9. Another exemplary tray is a multi-channel rehydration/electrophoresis tray of the invention, as described in detail below in connection with Figures 6-7B and 10A- 10B. The gel strip is then bathed in an aqueous rehydration buffer. It is typically desirable to cover the bathed gel strip so as to minimize evaporation during the rehydration. The buffers used in IEF often contain high concentrations of solute (even saturating concentrations). Those solutes, such as urea, may precipitate in response to evaporation of solvent from the system because their concentrations increase above saturation. In addition, any change in concentration of ingredients such as salts, may affect the separation conditions and therefore affect reproducibility of results. The rehydration buffer will generally contain a suitable amount of denaturant(s), detergents, and a mixture of one or more carrier ampholytes. Rehydration buffers generally contain few if any salts such as NaCI, KCI, sodium acetate, etc. An exemplary rehydration buffer comprises: 2M thiourea, 7M urea, 1% ASB14. Another exemplary rehydration buffer, called IPG Buffer, is available through Amersham Pharmacia Biotech. Rehydration buffers for ampholyne based IEF are also available. Rehydration is carried out for as long as necessary, and often for more than 6 hours, more than 8 hours, more than 10 hours, or more than 20 hours. During the lengthy rehydration process the gel strip is preferably covered to prevent evaporation of any of the fluids of the rehydration buffer and to avoid adsorption of fluids from the atmosphere, hi general, it is undesirable to use an oily substance to cover the rehydrating matrix. It is one aspect of the invention to provide for a solid cover for rehydrating gel strips.

Sample to be subjected to IEF is generally loaded onto the gel strip prior to electrophoresis. The sample may be loaded during rehydration or afterwards. When carrier ampholytes are being used, a pre-focusing step is performed before sample loading. One method of loading a sample is to use a sample cup. A cup is usually placed on top of the strip that was rehydrated in the absence of the sample. In the prior art, a non-polar fluid, such as mineral or silicone oil of suitable viscosity, then covers the strip. In the case of cup loading of proteins, the proteins enter the matrix through the interface between the matrix and the sunounding liquid. Generally, the cup is placed onto the matrix with appropriate pressure, and the sample is introduced into the cup and thus into contact with the matrix. The sample is then electrophoretically inserted from the cup into the strip. Depending on the application, the cup is either placed near either the acidic or basic end of the strip (alternatively, cathodic or anodic end). In one embodiment, the gel strip is covered by a suitable solid cover, such as made of glass, having at least one opening for cup loading of the sample.

Especially for the cup loading of proteins with membrane spanning domains, the invention offers the additional advantage that the proteins do not need to contact a non-polar fluid for which they may have high affinity.

In other embodiments, the sample is simply included in the rehydration or other fluid bathing the matrix. Thus, the sample subjected to IEF is loaded onto the gel strip prior to electrophoresis. WT en the gel strip is an IPG strip, the sample is often solubilized in the rehydration buffer. The rehydration buffer is then applied to the dry IPG strip using the method and device of the invention. Thus the sample is spatially spread/distributed over the entire strip volume.

It is a further advantage of the invention that the rehydration buffer can be applied to dry gel strips at spatially distinct locations. Especially, during the application of the rehydration buffer, the pipetor tip can be moved along the length of the gel strip. Such a rehydration buffer application leads to more uniformly wetting of the strip and improved rehydration. This mode of operation is beneficial for achieving rehydrated gel strips of more constant thickness.

Isoelectric focusing is generally accomplished by applying an electrical potential difference across a gel supporting a pH gradient. One of skill in the art will, in view of this specification, appreciate that there are many ways of doing so. One simple embodiment is to provide an electrical contact, or electrode, at either end of the gel supporting a pH gradient. Each electrical contact is then connected to a different pole on a power supply. Voltages to be applied will vary depending on conditions and the desired ranning time. Exemplary voltages include 100V, 200V, 300V, 400V, 500V, 600V, 1000V, 2000V, 3000N and 4000N. In certain embodiments, voltage is varied over time, typically linear ramping up to a certain plateau. In an exemplary embodiment, voltage is increased linearly from 300N to 3500N. In certain embodiments a combination of ramps and constant voltage sections are used. In certain embodiments the method and device of the invention provide for the use of mediating filters, placed between each electrode and the separation matrix. Mediating filters, such as filter papers, are essential for nanow range pH gradients, salt removal, electric field tempering, preventing and controlling matrix de-hydration and providing buffering compartments. The use of filters as buffering reservoirs is essential, especially when carrier ampholytes are utilized to generate pH gradients in the separation matrix.

In certain embodiments, temperature control systems are used to maintain the gel supporting a pH gradient at a constant temperature. For example, the electrophoresis unit may be contained in a refrigeration unit, or a liquid coolant may be circulated in close proximity to, but not in fluid contact with, the gel supporting a pH gradient.

IEF may be used as a stand-alone technique, in which case the gel supporting a pH gradient will typically be removed from the electrophoresis apparatus and subjected to a detection method. In general, the detection method to be performed will be determined by the experimenter. Exemplary detection methods may include protein stains, such as Coomassie blue, Amido black, silver staining, Ponceau S staining. Detection methods may include light absorbtion spectroscopy, such as UV absorbtion and/or visible light absorbtion and/or densitometry and/or colorimetry and or fluorimetry, etc studies. Portions of the gel supporting a pH gradient may be dissected and any sample therein removed by, for example, electroelution or dialysis. Removed sample may be subjected to analysis by mass spectrometry, optionally coupled with a protease digestion step, etc. A direct MALDI-TOF analysis could be performed from the gel supporting a pH gradient.

Often, IEF will be used in combination with an additional resolution step.

For example, the isoelectrically focused sample may be used in a second resolution step that resolves the sample components by molecular size. Such a step may be a denaturing polyacrylamide gel electrophoresis, in which case the combined resolution steps are termed a 2D-gel electrophoresis. In general, as will be understood by one of skill in the art in view of this specification, any of the method steps described herein may be combined or interconnected in various ways to give methods tailored to particular applications.

IEF Apparatus

An exemplary embodiment of an apparatus 10 for conducting isoelectric focusing according to the present invention is illustrated in Figures 1-4. The apparatus 10 includes a tray 12 for receiving a gel strip 14 and a cover 16 that may be positioned over the tray 12 to cover all of the channel 20 and all or a portion of the tray 12. The apparatus 10 is particularly suited for perfonning IEF procedures in the absence of a cover layer of hydrophobic fluids in accordance with the IEF methods disclosed herein, hi addition, the apparatus 10 permits the performance of IEF procedures with a buffer that is weakly conductive and, often, aqueous. In certain embodiments, the apparatus 10 is reusable, i further embodiments, the apparatus 10 permits the detachment and removal of cover 16 from the tray 12 during the performance of any of the steps of the IEF procedure.

The tray 12 of the exemplary apparatus 10 is generally block-shaped, although other shapes are possible, and includes a top surface 18. A channel 20 is formed on the top surface 18 to receive the gel strip 14 during IEF, rehydration or other processing. The channel 20 includes a first end 22 spaced apart from a second end 24. The chaimel 20 is preferably sized and shaped to receive a gel strip. In one embodiment, the length L and the width W of the channel may be selected to generally approximate the length and width, respectively, of the gel strip while also providing sufficient clearance to permit the gel strip to be positioned within the channel and to be removed from the channel. In this manner, the clearance between the gel strip and walls of the channel may be minimized, hi a preferred embodiment, the depth D of the channel may be selected to generally approximate the height of the rehydrated gel strip, when positioned in the channel, so that the top and bottom surfaces of the gel strip touch the entire bottom surface of the chamiel and the entire bottom surface 26 of the cover 16 when the cover is positioned on the top surface 18 of the tray 12. For example, in the case of a 3.5-10.0 NL dry gel strip from Amersham Biosciences having a length of 18 cm, a width of 3 mm, and a depth of approximately 0.2 mm, the channel may be sized as follows:

Chamiel Length (L): 18 - 25 cm

Channel Width (W): 3 - 5 mm Channel Depth (D): 0.5 - 1.2 mm

The shape of the channel 20 is preferably selected to match the shape of a gel strip. For example, in the case of a gel strip having a rectangular cross section, the channel 20 is also preferably rectangular in cross-section.

In an alternative embodiment the channel cross section may be non- rectangular and shaped to allow suitable fluid meniscus pinning. In this manner, when rehydration buffer is applied to a dry gel strip, spilling of the rehydration buffer can be minimized. For example, a suitable radius or a concave chamfer may be used, as is known in the art. In additional embodiments the cross section of the channel may be varying, as a method to control the dimensions and density of the matrix as well as to control the electric field at selected spatial locations. A specific embodiment is shown if Figure 8.

The tray 12 may be constructed from glass, plastic or other non-conducting and chemically inert materials suitable for use in diagnostic or medical equipment. The tray 12 may be machined to form the channel 20. Alternatively, the tray 12 and the channel 20 may be formed through a molding process. Other manufacturing processes may be used to construct the tray 12 and the channel 20 without departing from the scope of the present invention.

The cover 16 of the exemplary apparatus 10 may include a first electrode compartment 32 and a second electrode compartment 34 formed in a bottom surface 26 of the cover 16. The first elecfrode compartment 32 and the second electrode compartment 34 each contain an electrophoresis electrode E that may be connected to a power supply to effect IEF processing. Two pairs of conduits 28 are provided in the cover 16 to permit the electrophoresis electrodes E to be connected to the power supply by a wire or other conductive medium housed in the conduits 28. The conduits 28 may extend from each elecfrode compartment 32, 34 to the top surface 30 of the cover 16. The number of conduits 28 as well as the arrangement of the conduits 28 may be varied without departing from the scope of the present invention. For example, a single conduit may be used for each electrode compartment or the conduits 28 may be arranged to connect the electrode compartment to a surface of the cover 16 other than the top surface 30. Furthermore, electrical contacting may also be in the form of spring contacts.

The first electrode compartment 32 and the second electrode compartment 34 may be positioned on the cover 16 such that the first electrode compartment 32 overlies the first end 22 of the channel 20 and the second electrode compartment 34 overlies the second end 24 of the channel 20 when the cover 16 is positioned over the tray 18. As best illustrated in Figure 3, the first electrode compartment 32 is thus spaced apart from the second electrode compartment 34 a distance S substantially equal to the length L of the channel 20. In this manner, the electrodes E can be placed into contact with the ends of the gel strip during IEF so that IEF takes place in between the electrodes, hi particular, it is desirable to maximize the useful pH range of the gel strip, therefore, the electrode compartments 32 and 34 preferably overly the respective ends 22 and 24 of the channel in order to contact the gel strip positioned in the channel near its ends. The width W_E of each electrode compartment 32, 34 is preferably substantially equal to or greater than the width W of the channel 20 to provide electrode contact across the entire width of the gel strip during IEF. A suitable filter paper or other conductive medium optionally may be positioned in each electrode compartment 32, 34 to insure electrical contact between the electrodes E and the gel strip. The depth of each electrode compartment 32, 34 may be selected to accommodate the electrode E and, if used, the filter paper. The depth may be selected to optimize the contact pressure of the electrode and/or filter paper on the gel. Suitable, controlled, contact pressure can also be created by the use of spring electrodes, if used. In a preferred embodiment, the depth may be selected so that when the filter paper is contacting the electrode in the electrode compartment, the surface of the filter paper contacting the gel strip is flush with the bottom surface 26 of the cover. The bottom surface 26 of the cover 16 is preferably sized and shaped to engage the top surface 18 of the tray 12 in a sealing relationship to substantially inhibit the flow of fluid to and from the channel 20 when the cover 16 is positioned over the tray 18. As best illustrated in Figures 1 and 3, for example, the bottom surface 26 of the cover 16 preferably has a length and width greater than the length L and the width W of the channel 20 such that the bottom surface 26 overlaps the channel 20 when the bottom surface 26 and the top surface 18 are in contact. Moreover, the portion of the bottom surface 26 between the first electrode compartment 32 and the second electrode compartment 34 may also substantially overlie and, thus, cover the chaimel 20. h this manner, the bottom surface 26 of the cover 16 substantially covers the channel 20 with the exception of the two electrode compartments 32, 34 positioned at either end of the channel 20. The cover 16 optionally may be clamped into contact with tray 12 to facilitate electrical contact between the electrodes E and the gel strip as well as to assure sealing.

Optionally, the first and second electrode compartments, 32 and 34, may contain small holes to permit the escape of electrolysis product gases produces in the vicinity of the electrodes E (not shown). Such holes should be dimensioned as to permit electrolysis gases to escape, yet minimize the strip drying during the electrophoresis process. Alternatively, the first and second electrode compartments 32 and 34 may be sized to function as storage compartments for the generated electrolysis gases during electrophoresis, away from the location of the IEF process in the strip.

Although the cover 16 of the exemplary apparatus 10 is illustrated as being of similar size and shape to the tray 12, one skilled in the art will appreciate that the cover 16 may be of any size or shape suitable to provide a sealing relationship between the bottom surface 26 of the cover 16 and the top surface of the tray 12. Preferably, the bottom surface 26 of the cover 16 is complementary in shape to the top surface 18 of the tray 12 to facilitate a sealing relationship between the surfaces. Complementary shapes are also beneficial to accurately reposition the cover on the tray when the cover is rejoined after detachment during any of the steps of IEF. For example, in the case of a planar top surface 18, the bottom surface 26 is preferably also planar in shape. In a further example, in the case of a top surface 18 having a convex shape, the bottom surface 26 is preferably concave in shape. Suitable alignment marks or alignment systems optionally may be provided on the top surface 18 and/or the bottom surface 26 to facilitate proper alignment of the cover 16 with the tray 18. Exemplary alignment systems may include one or more notches, grooves, hinges or other indents formed on either the top surface 18 of the tray 12 or the bottom surface 26 of the cover 16 and one or more complementary protrusions formed in the mating surface. Furthermore, suitably featured mating surfaces may be used to prevent relative motion of the surfaces upon contacting.

A suitable sealing mechanism can be added to seal both surfaces together.

Preferably, such a mechanism is external to the fray and the cover.

To achieve good sealing the cover and/or the tray may have fritted surfaces of suitable roughnesses. Alternatively sufficiently smooth surfaces, such as made of glass or acrylic plastic can be used.

The cover 16 may be constructed from glass, plastic or other materials suitable for diagnostic or medical equipment. The cover 16 need not be the same material as the tray 12. Additionally, selective coatings optionally may be applied to selected surfaces of the cover 16 and the tray 12, including the channel 20, to facilitate the IEF process. For example, selected surfaces, such as the bottom surface 26 of the cover 16 or the surfaces of the channel 20, may be silanized or coated with a suitable polymer (thin layer or monolayers). Suitable surface coatings, such as silanization, may be beneficial to specifically tailor endosmotic flow behavior or surface interactions with the separation medium or proteins. A specific example of a surface coating agent is Plus-One™ Repel-Silane ES sold by Amersham Biosciences. The cover 16 and/or the tray 12 optionally may be constructed of a transparent or semi-transparent material to allow viewing of the gel strip during processing.

The tray 12 and/or the cover 16 of the apparatus 10 optionally may be provided with a temperature control system for controlling the temperature of the gel strip during processing. The tray 12 of the apparatus 10 may also be used to rehydrate gel strip by supplying the channel 20 with a suitable fluid for rehydration, such as a rehydration buffer, and covering the tray 10 with a plate 50 of glass or other material, as best illustrated in Figure 5. The plate 50 is preferably sized and shaped to engage the top surface 18 of the tray 12 in a sealing relationship. The depth D of channel 20 is preferably selected for depth such that when a dry gel strip is rehydrated in the channel, without the use of oil or any other hydrophobic cover fluid, the top surface of the gel strip entirely contacts the mating surface of plate 50. The plate 50 optionally may be provided with an opening for inserting a pipette or other instrument to deliver the rehydration or other solution to the channel 20 while covered. A second opening may also be provided to allow air to escape the channel 20 as the channel 20 is filled. In one embodiment, the opening(s) may be self- sealing to permit the opening to close after insertion of the pipette. A resilient, puncturable membrane may be positioned within the opening to provide self-sealing. Alternatively, a closure element may be provided to close the opening(s) when not in use. One or more analogous openings optionally may also be provided in the cover 16. In certain embodiments, the cover 16 and plate 50 are interchangeable and can be detached from and/or repositioned onto tray 12. In another embodiment, rehydration can also be performed with detachable cover 16. The apparatus 10 permits the detachment, removal and replacement of cover 16 or plate 50 from the tray 12 during and between any of the rehydration steps and the electrophoresis steps of the IEF procedure.

Figures 6 illustrates alternative embodiment of a tray 112 for conducting high throughput IEF according to the present invention. A tray 112 having multiple parallel chaimels 20 for receiving multiple gel strips is illustrated in Figure 6. The tray 112 allows multiple IEF procedures to be conducted simultaneously. The channels 20 can be arranged in parallel as illustrated, or can be arranged in other configurations depending on the space available on the tray 112 and the size and shape of the gel strips employed. Likewise, the number of channels 20 can be varied depending on the space available on the tray 112. Figures 7 A and 7B illustrate alternative embodiments of a cover 116' and 116" for use with a multi-channel tray, such as, for example, the multi-channel tray 112 illustrated in Figure 6. The cover 116' provides a pair of generally parallel electrode compartments 132' and 134' that extend along the width of the tray 116'. Each electrode compartment 132' and 134' may house an electrode E for effecting IEF. The electrode compartments 132' and 134' are sized and positioned overlie the ends of each of the channels 20 of a multi-channel tray, such as the tray 112 illustrated in Figure 6. hi this arrangement, each electrode E may be in contact with each gel strip positioned in the multi-channel tray, possibly through a suitable filter paper. In certain cases it is desirable to have the electrode E not flush with the surface of the cover. Such a recess permits the insertion of a suitable filter paper in the electrode compartment between the electrode E and the gel strips. The dimensions of the recess should be selected so that upon sealing the tray and the cover, the pressure on the gel strip is optimal. In a preferred embodiment the depth of the recess is selected so that when a filter paper of suitable size is positioned in the recess, one surface of the filter paper is in contact with the electrode in the electrode compartment and another surface of the filter paper is flush with the bottom surface of the cover. In this manner, the surface of the cover and the filter paper apply equal pressure on the gel strip. Furthermore, it may be advantageous to provide for electrode compartments 132' and 134' with recesses only substantially at the locations overlying channels 20 of a multi-channel tray 112. Furthermore, the recess may be dimensioned to provide a tight fit for a wetted filter paper, preventing the filter paper dropping out. Additionally, this may be achieved by having a recess of varying (e.g concave) cross section.

Referring to Figure 7B, the cover 116" includes a plurality of spaced apart electrode compartments 132" and 134" extending along the width of the cover 116". Each of the plurality of electrode compartments houses a separate electrode E'. Each electrode compartment is sized and positioned to overlie an end of one of the channels 20 of a multi-channel tray, such as tray 112 of Figure 6. In both arrangements, IEF procedures may be conducted within selected channels or all the channels of the multi-channel tray. Furthermore, each of the individual channels may be selected for length, width or depth. The pairs of electrodes formed by each of the individual electrodes 132" and 134" maybe contacted to a single or to multiple power supplies.

A tray of another embodiment to perform the method of the invention is shown in Fig. 8. The tray 12 is generally block-shaped, although other shapes are possible, and includes a top surface 18. A channel 20' consisting of a long first subunit of length L ending in a tapered second subunit of length L' and yielding a trombone-like stmcture or silhouette, is formed in the top surface 18 to receive the gel strip 14 during IEF, rehydration or other processing. The channel has a long constant cross section part and a short monotonous diverging part, having a common nanow cross section of width W, the short part diverging to a width W' . The long part of length L is used to separate the bio-molecules, whereas the short part serves to smoothly taper out the end conditions of the long part. In particular, the tapered second subunit is at the cathodic side of an electrophoresis device. For example, W may be chosen 3 - 5 mm, L 13 - 25 cm, W' 5 — 20 mm and L' 3 - 8 cm. Preferably, channel 20' contains a medium wherein the medium is a tapered immobiline strip or ampholine strip. The end taper is advantageous because it reduces the electric field strength near the cathodic end of the electrophoresis minimizing cathodic drift effects and other disturbances, potentially increasing the operative pH span of the ampholine and immobiline strips, hi an additional embodiment, a commercial gel strip, such as can be purchased from Amersham Biosciences, may be connected with a tapered gel (e.g. agarose or acrylamide) of suitable pH and ionic strength. These gels can be connected together either by physical contacting or by sealing them together as is known to those skilled in the art. It is preferable that the region of contact be of electrical resistance lower than that of the gels to avoid the creation of "hot spots". In an additional embodiment, the channel of width W has a taper from both its ends, either with identical tapers or not. A suitable cover for the tray of Figure 8 has two electrode compartments separated by a distance S substantially equal to L + L' with the width W_E of one electrode compartment less but approximating W and the width of the other electrode compartment W_E> less but approximating W'. In Figure 9 a perspective view of yet another embodiment of the apparatus is shown. The tray 12 of the exemplary apparatus 10 is generally block shaped, although other shapes are possible, and includes a top surface 18. A channel 20" of depth D2 is formed in the top surface 18 to receive the gel strip 14 during IEF, rehydration or other processing. The cover 16 of the apparatus 10 may be positioned on the tray 12 in a sealing relationship as indicated by the arrows. Preferably, the bottom surface 26 of the cover 16 has a protrusion 23 of thickness Dl. Protrusion 23 preferably has dimensions substantially equal to the width and length of the channel 20". The thickness Dl of the protrusion 23 and the depth D2 of the channel 20 are specifically selected to ensure that the fully rehydrated gel strip, when deposited in the channel 20", contacts, along its entire length, the bottom surface 26' of the protrusion 23 of the cover 16. hi addition, protmsion 26' may also contain suitable electrode compartments, for use with or without filters between the electrode and the gel strip. This embodiment is advantageous because channels of sufficient depth dimmish the possibility of rehydration buffer spilling when rehydration buffer is applied during rehydration. In an additional prefened embodiment, the bottom surface 26' of the protrusion 23 is coated with a suitable surface coating, such as a silanization. In addition, protrusion 23 is complementary to channel 20". Therefore, the cover can easily be repositioned on the tray during any of the steps of the IEF process.

Figure 10A and 10B illustrate practical embodiments of the device of the invention. The embodiment shown in Figure 10A comprises a mechanical assembly 160. The mechanical assembly has a first part 161 for receiving a tray, such as multichannel tray 112 of the embodiment. Certain features on the top surface of the first part of the mechanical assembly 161 mate complementary features on the bottom surface of tray 112. Therefore, once tray 112 is positioned on the first part of the mechanical assembly 161, the fray is immobilized with respect to the first part of the mechanical assembly 161. The mechanical assembly also has a second part 162 for receiving exemplary cover 116'. Interchangeable cover 116' can be slid in and out laterally during any of the steps of the isoelectric focusing process. Cover 116' is held in place by holders 167 and by a locking mechanism (not shown) that fully immobilizes cover 116' with respect to the second part of the mechanical assembly 162. Hinges 165 connect the first part of the mechanical assembly 161 to the second part of the mechanical assembly 162. Sealing of tray 112 with the cover 116' can therefore be performed without any lateral movement. Furthermore, mechanically assembly 160 permits the accurate repositioning of the cover on the tray. In an alternative embodiment, a cover 116" including a plurality of spaced apart electrode compartments 132" and 134" extending along the width of the cover 116", as shown in Figure 10B is used. With this embodiment it is possible to power each strip individually. In a further embodiment, either cover 116' or cover 116' is replaced by a cover without electrodes, such as plate 50 of Figure 5.

In yet another embodiment, multiple channels 20' may be formed in the tray

12.

One skilled in the art will appreciate that apparatuses described herein are not limited to use in IEF processing but may be used in other testing, analyzing, or treatment procedures without departing from the scope of the present invention. Specifically, while throughout the disclosure many embodiments are presented with gel strips, those of skill in the art will readily appreciate how to adapt this to isoelectric focusing based on e.g. carrier ampholytes.

The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

Example

Exemplary rehydration buffers may be prepared as follows:

Rehydration buffer (lx) (2M thiourea, 7M urea, 1% ASB14) Place in a 50 ml tube the following ingredients, solubilize all except thiourea; add thiourea last.

Final concentration Amount

Urea (FW 60.06) 7 M 4.2042 g Thiourea (TW 16,2) 2 M 1.5224 g

ASB14 (20% stock) 1% (w/v) 0.5 ml

Double distilled H₂0 to 10 ml. 4 ml

Place at 32°C on a rotating table for 30 minutes, until dissolved. Add 0.1 g amberlite and rotate below 32°C for 10 minutes. Measure volume. Add ddH₂0 to 10 ml if necessary.

Pass 10 ml ddH 0 through a 0.45 syringe filter and filter the rehydration solution next

Store in 1 ml aliquots at -70°C

1. Just prior to use, slowly thaw an aliquot or take the required volume of rehydration solution.

2. Add IPG Buffer (100%): Add IPG buffer to a final concentration of 2% (i.e., 20 μl IPG buffer for 1 ml rehydration solution).

3. Prepare IM TCEP-HCL in ddH 0, divide to 50 μl aliquot and freeze at -20°C. Avoid repeated freeze thaw, after the second thaw throw the aliquot. Add TCEP to 5mM final concentration in rehydration solution (i.e., the stock concentration is x200). 4. Add protease inhibitor cocktail 3 (x200 stock).

5. Sample addition. The sample can either be a protein pellet or in solution. Pelleted sample is resuspended directly in lx rehydration solution containing the ingredients as specified above. For example, 150 μg plasma membrane proteins of concentration 2.4 μg/ μl may be solubilized to a total of 330 μl rehydration buffer for 13 cm IPG strips.

6. Incubate sample in rehydration solution for 3 hours. Spin at 14, 000 rpm for 30 minutes at 25°C and transfer supernatant to a fresh tube.

Gel strip rehydration (with oil cover)

For gel strip rehydration, a multi-channel tray having a plurality of channels, such as the multi-channel tray described above, may be employed. The tray may be selected based on the length of the gel strips used. Rehydration may be performed in accordance with the following exemplary steps:

A. Prepare the multi-channel tray by cleaning and drying the tray.

B. Apply the rehydration solution to the channels of the tray by, for example, pipetting the appropriate volume of sample containing rehydration solution into each channel.

Gel strip length (cm) Total volume per strip (μl )

7 178

11 280

13 330

18 450

24 610

C. Place a gel strip within the channel of each tray by removing the protective cover from the gel strip and positioning the gel strip with the gel side up into the rehydration solution. To help coat the entire gel strip, gently lift and lower the strip and slide it back and forth along the surface of the solution.

D. Overlay the gel strips with 2 ml cover fluid (such as PlusOne DryStrip Cover Fluid by Amersham Biosciences) and close the multichannel tray with a cover, such as the glass plate described above.

E. Allow the gel strips to rehydrate preferably at room temperature. A minimum of 10 hours may be necessary for rehydration. If no urea is added to the rehydration solution, 4 hours of reswelling may be sufficient.

Gel strip rehydration (without oil cover): □ Use the glass IPG device for rehydration. Level the tray to ensure horizontality.

□ Put the gel strips face-up and then cover them with 300 μl rehydration buffer each. □ Leave the cover open for 1 h to allow initial rehydration to prevent spilling the liquid upon closing.

□ Close the cover and rehydrate overnight.

Preparing for electrophoresis

The following exemplary steps may be followed to prepare for electrophoresis:

□ Clean the glass tray with soap and water, then with ethanol. Siliconize the cover with PlusOne Repel-Silane.

□ Set up the cooling system to 20°C. Q Remove the strips from the tray.

□ Rinse each strip using about 2 ml ddH₂O (to remove chaotrope crystals).

□ Put the strips on Wattmann paper to remove excess water.

□ Put the strips back into their respective channels. □ Cut two electrode filter paper strips long enough to cover the strips.

□ Wet each filter paper with 46 μl ddxH₂O for each cm and remove water excess between two Wattmann sheets.

□ Put the electrodes across the two ends of the strips. Close the cover ensuring good contact between the cover and the tray. □ Connect the power supply (Multiphor II - EPS 3501 XL, Amersham Biosciences)

□ Run the following program:

1. 0 to 200 N ramped up in 5 min.

2. 200 N l h. 3. 200 to 500 N ramped up in 1 h.

4. 500 to 1000 N ramped up in 30 min.

5. 1000 to 3500 N ramped up in 1 h.

6. 3500 N 3 h.

Second dimension separation (SDS-PAGE):

Materials (Stock solutions):

Resolving gel: 30% Acrylamide/Bis (30% T / 2.6% C) (Biorad Laboratories).

Dissolve 91 g Tris base in 300 ml ddH₂O. Adjust to pH 8.8 with IN HCl (43 ml concentrated HCl and 457 ml ddH₂O). Add ddH₂O to 500 ml total volume. Filter the solution through a 0.45 μm filter. Store at 4°C up to 1 month.

Running buffer flOx : Tris-glycine-SDS (250 mM-1.98 M-1% w/v) pH 8.3 (Biorad Laboratories) Make a fresh dilution for each run. Make 5L and add 25 ml 20% SDS.

APS (40%): To prepare 10 ml of the solution, dissolve 4 g ammonium persulfate in 10 ml ddH₂O. Make 0.5 ml aliquot and freeze at -20°C.

Set up the gradient maker and prepare gel solutions:

1. Place a magnetic stirrer under the gradient maker. Connect the outlet valve of the gradient maker to a pump and to the end of the flexible tubing insert a narrower non-flexible tubing (~4 cm). Attach to the end of the tubing a micropipet tip.

2. Place a stir bar into the mixing chamber of the gradient maker (the chamber connected to the outlet).

3. Set up the gel cassette and place at 4°C for at least 15 minutes prior to gel casting. 4. Prepare heavy and light acrylamide solutions:

Gel dimensions: 160x180x1.5 mm (Hoeffer Instrument)

5. With the outlet port and interconnecting valve between the two chambers closed, pipet 17 or 20 ml (depending on the plates, see step 4) of light acrylamide gel solution into the reservoir chamber (the chamber which is not near the outlet) for one gel.

6. Open the interconnecting valve briefly to allow a small amount (-200 μl) of light solution to flow through the valve and into the mixing chamber. Remove the small amount of the light solution that flowed into the mixing chamber back into the reservoir chamber.

7. Add 17 or 20 ml heavy solution into the mixing chamber.

8. Add 5.6 μl TEMED for 17 ml acrylamide solution (or 6.6 μl for 20 ml, 4.6 μl/14ml) into each chamber. Mix the solutions with a 10 ml pipet.

9. Place the cooled gel cassette beside the gradient maker. Insert the tip between the two glass plates.

10. Start stirring the stir bar in the mixing chamber. Add 13.5 μl 40% APS to 17 ml acrylamide solution (16 μl to 20 ml acrylamide solution, 11 μl/14 ml) to both chambers. Add first to the light solution, mix by 10 ml pipet, and than add to the mixing chamber.

11. Open the interconnecting valve completely.

12. Open the outlet to the gradient maker slowly. Start the pump at a setting of 7.5.

13. Overlay the gradient gel with 400 μl double distilled water with a 20- 100 μl pipetman. After ~ 2 hours add carefully lxTris-Glycine to top the glass to prevent drying overnight. Cover the gel with saran-wrap and keep at room temperature overnight.

Running conditions

Set up a tank with lx running buffer at a temperature of 15°C. Add SDS to 0.2 % in the lx running buffer (it contains 0.1%), i.e., 25 ml 20% SDS to 5L lx running buffer. 1. 10 mA/gel, 100N, 5W/gel for 15 minutes.

2. 20 mA/gel, 150N, 5W/gel for 10 hours.

3. 5 mA/gel, 15 ON, 5W/gel until the gel is removed.

4. Run until the bromophenol blue has migrated off the lower part of the gel.

Stain gels as is known the art, e.g. silver staining.

Incorporation by Reference

All of the patents and publications cited herein are hereby incorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the solution, 11 μl/14 ml) to both chambers. Add first to the light solution, mix by 10 ml pipet, and than add to the mixing chamber.

11. Open the interconnecting valve completely.

Running conditions

Set up a tank with lx running buffer at a temperature of 15°C. Add SDS to 0.2 % in the lx running buffer (it contains 0.1%), i.e., 25 ml 20% SDS to 5L lx running buffer. 1. 10 mA/gel, 100N, 5 W/gel for 15 minutes.

2. 20 mA gel, 150N, 5W/gel for 10 hours.

3. 5 mA/gel, 150N, 5 W/gel until the gel is removed.

4. Run until the bromophenol blue has migrated off the lower part of the gel. Stain gels as is known the art, e.g. silver staining.

Incorporation by Reference

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by any claims drafted later on based on the disclosure herein.

Claims

What is claimed is:

1. An apparatus for conducting isoelectric focusing, electrophoresis and/or rehydration, the apparatus comprising

(a) a tray having a first surface and at least one channel formed in the first surface, the channel having a length, width, depth and a floor and being sized and shaped to receive a rehydrated gel strip having a first and second surface and wherein the length and width of the channel is substantially equal to the length and width of the gel strip;

(b) a cover having a first surface and at least one first electrode compartment and at least one second electrode compartment formed in the first surface, the at least one first electrode compartment and the at least one second electrode compartment being spaced apart on the first surface of the cover a distance substantially equal to the length of the channel, wherein the first surface of the cover is sized and shaped as to mate the first surface of the tray substantially sealing the chamiel formed in the first surface of the tray to form an elongated enclosure, and

(c) a mechanical assembly

- for receiving the fray and the cover comprising a means for mating the first surface of the tray to the first surface of the cover, and

- whereby the first surface of the cover can be disjoined and rejoined in a sealing relationship with the first surface of the tray, wherein when the gel strip is positioned on the floor of the channel and the first surface of the cover is mating the channel in a sealing relationship, the entire first surface of the gel strip is in close contact with the floor of the channel and the entire second surface of the gel strip is in close contact with the first surface of the cover, except for those portions of the first surface of the cover containing the first and second electrode compartments.

2. An apparatus for conducting isoelectric focusing electrophoresis and/or rehydration, the apparatus comprising,

(a) a tray having a first surface and at least one channel formed in the first surface, the channel having a length, width, depth and a floor and being sized and shaped to receive a rehydrated gel strip having a first and a second surface, and wherein the length and width of the channel is substantially equal to the length and width of the gel strip and wherein at least one first electrode compartment and at least one second electrode compartment are formed in the floor of each of the at least one channel, the at least one first electrode compartment and the at least one second electrode compartment being spaced apart on the floor of the at least one channel a distance substantially equal to the length of the channel; and

(b) a cover having a first surface sized and shaped as to mate the first surface of the tray substantially sealing the channel formed in the first surface of the tray to form an elongated enclosure; and

(c) a mechanical assembly

- for receiving the tray and the cover comprising a means for mating the first surface of the tray to the first surface of the cover, and

- whereby the first surface of the cover can be disjoined and rejoined in a sealing relationship with the first surface of the tray, wherein when the gel strip is positioned on the floor of the channel and the first surface of the cover is mating the channel in a sealing relationship, the entire first surface of the gel strip is in close contact with the floor of the channel, except for those portions of the floor of the channel containing the first and second electrodes, and the entire second surface of the gel strip is in close contact with the first surface of the cover. 3. An apparatus for conducting rehydration of an immobilized pH gradient gel strip, the apparatus comprising, (a) a tray having a first surface and at least one channel formed in the first surface, the channel having a length, width, depth and a floor and being sized and shaped to receive the gel strip and rehydration buffer, and wherein the length and width of the channel is substantially equal to the length and width of the gel strip, and

(b) a cover for the tray having a first surface sized and shaped as to mate the first surface of the tray sealing the channel formed in the first surface of the tray to form an elongated enclosure, and

(c) a mechanical assembly - for receiving the tray and the cover comprising a means for mating the first surface of the tray to the first surface of the cover, and whereby the first surface of the cover can be disjoined and rejoined in a sealing relationship with the first surface of the tray, wherein when the gel strip is positioned on the floor of the channel and the first surface of the cover is mating the channel in a sealing relationship, and when the gel strip is rehydrated, the entire first surface of the gel strip is in close contact with the floor of the channel and the entire second surface of the gel strip is in close contact with the first surface of the cover.

4. The apparatus of any of claims 1 to 3 wherein the distance between the floor of the channel and the first surface of the cover is less than the thickness of the gel strip when rehydrated, and wherein the distance between the floor of the channel and the first surface of the cover permits the rehydration of the gel strip to at least 80% of its thickness when rehydrated.

5. The apparatus of any of claims 1 to 3 wherein the cross section of the channel is spatially varying.

6. The apparatus of claim 5 wherein the at least one end of the channel is tapered.

7. A method for isoelectric focusing separation comprising: (a) providing a gel strip supporting a pH gradient during isoelectric focusing electrophoresis, wherein the gel has a length, width, height, and a first surface and a second surface both defined by the length and width of the gel; (b) placing said gel strip on the floor of a channel formed in a tray;

(c) providing a solid detachable cover for the channel, wherein the cover and/or the floor of the channel comprises a first and second electrode compartment;

(d) covering the gel strip with said cover, wherein when the gel strip is positioned on the floor of the channel and the cover is mating the tray in a sealing relationship, the entire first surface of the gel strip is touching the floor of the channel and the entire second surface of the gel strip is touching the cover, except at the first and second electrode compartments, wherein the electrodes contact the surface of the gel strip directly or by a mediating filter

(e) loading a sample onto the gel strip before, after or concurrently with any of parts (a) - (d)

(f) applying an electrical field across the gel strip so as to obtain an isoelectrically focused sample. 8. The method of claim 7, further comprising a rehydration process.

9. The method of claim 8, wherein the rehydration process comprises:

(a) providing a dry gel strip

(b) placing said gel strip on the floor of a channel formed in a tray

(c) bathing said gel strip with a rehydration buffer (d) providing a solid detachable cover for the channel

(e) covering the gel strip and rehydration buffer with said cover in a sealing relationship, wherein when the gel strip is rehydrated, the entire first surface of the gel strip is touching the floor of the channel and the entire second surface of the gel strip is touching the cover, except for any electrode compartments, if applicable.

10. The method of claim 8, wherein at the completion of the rehydration process the gel is rinsed.

11. The method of any of claims 7 to 10, wherein the detachable cover can be disjoined and rejoined in a sealing relationship with the tray.

12. The method of any of claims 7 to 10, wherein the cover for the tray in contact with the gel is coated. 13. The method of claim 12, wherein the coating is a silanization.

14. The method of claim 12, wherein the coating is hydrophilic.

15. The method of any of claims 7 to 10 wherein crystallites remaining after rehydration are rinsed away.

16. The method of any of claim 7 to 10 wherein the sample comprises proteins of a category selected from the group consisting of: membrane proteins, hydrophobic proteins, protein having one or more lipophilic regions when denatured, proteins difficult to solubilize in aqueous environments, proteins solubilized by high concentrations of chaotropes and proteins solubilized with detergents. 17. The method of claim 16, wherein the rehydration buffer contains chaotropes in concentrations sufficient to solubilize proteins that are otherwise difficult to solubilize.

18The method of claim 16, wherein the rehydration buffer contains urea and thiourea. 19. The method of any of claims 7 to 10, wherein the rehydration buffer contains urea and thiourea.

20. The method of any of claims 7 to 10 wherein the cathode is applied at the tapered end of the channel.

21. The apparatus of any of claims 1 to 3, wherein the gel strip is selected from the group consisting of: a gel strip containing an immobilized pH gradient and a gel strip containing two or more ampholytes.

22. The apparatus of any of claims 1 to 3, wherein the gel strip comprises a support that forms the first or second surface of the gel strip.