WO2004020991A1

WO2004020991A1 - An electrophoresis gel having improved swelling properties

Info

Publication number: WO2004020991A1
Application number: PCT/AU2003/001122
Authority: WO
Inventors: Benjamin Ross Herbert; Maurizio Bruschi; Luca Musante; Giovanni Candiano
Original assignee: Proteome Systems Intellectual Property Pty Ltd
Priority date: 2002-09-02
Filing date: 2003-09-01
Publication date: 2004-03-11
Also published as: AU2002951153A0

Abstract

The present invention provides an immobilised pH gradient (IPG) gel that is more uniformly reswollen than currently available gels. Furthermore, the improved IPG gel of the present invention can be produced with larger pore sizes than their conventional equivalents. Accordingly, in its broadest aspect, the present invention provides an immobilised pH gradient (IPG) gel comprising an organic acid or organic base, wherein when hydrated, the IPG gel is substantially uniformly swollen along the pH gradient of the gel.

Description

"An electrophoresis gel having improved swelling properties"

Field of the Invention

The present invention relates to an improved gel for electrophoresis and to a method of preparing an improved gel.

Background of the invention General Information

Unless the context requires otherwise or specifically stated to the contrary, integers, steps, or elements of the invention recited herein as singular integers, steps or elements clearly encompass both singular and plural forms of the recited integers, steps or elements.

Throughout this specification, unless the context requires otherwise, the word

"comprise", or variations such¹ as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The present invention is not to be limited in scope by the specific examples described herein. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

All the references cited in this application are specifically incorporated by reference herein.

The present invention is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, polymer science, protein biochemistry, and protein separation as described in, for example,

1. Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III; 2 Ferdinand Rodriguez: Principles of Polymer Systems, McGraw-Hill Publishing

Company Ltd, New Delhi, T M H Edition (1974)

3 Bjellqvist, B., Ek, K., Righetti, P.G., Gianazza, E., Gδrg, A., Westermeier, R. and Postel, W., J. Biochem. Biophys. Methods 1982, 6, 317-339. 4 Righetti, P.G., Immobilized pH Gradients: Theory and Methodology. Elsevier,

Amsterdam, 1990.

5 Righetti, P.G., Stoyanov, A., Zhukov, M., The Proteome Revisited: Theory and

Practice of All Relevant Electrophoretic Steps, Elsevier, Amsterdam, 2001.

6 Ek, K., Bjellqvist, B., Righetti, P.G., J. Biochem. Biophys. Methods 1983, 8,

134-155.

Righetti, P.G., Gelfi, C., J. Biochem. Biophys. Methods 1984, 9, 103-119

Background of the related art In the field of analysing macromolecules, one-dimensional and two-dimensional gel electrophoresis have become standard tools for separating and visualising macromolecules. The highest resolution method for separating macromolecules is two- dimensional electrophoresis. Such two-dimensional electrophoresis usually involves sequential separations in a first dimension by isoelectric focusing and in a second dimension by SDS gel electrophoresis. The standard method of performing the first dimension is isoelectric focusing using an immobilised pH gradient (IPG).

These immobilised pH gradients are polyacrylamide gels in which buffering groups responsible for the formation of the pH gradient are acrylamide derivatives co- polymerised into the gel with acrylamide and a cross-linker. These derivatives are called "Immobilines" by the manufacturer, Pharmacia. A gradient of these "Immobiline" groups is cross-linked to polyacrylamide. The polyacrylamide of commercial IPGs are cross-linked by bis-acrylamide or piperazine di-acrylamide (PDA) and often have an acrylamide concentration of 4%T.

The polyacrylamide gels are attached to an activated backing sheet, dried and cut into strips, approximately 3mm wide. In use, the dry strips are rehydrated in a protein solution.

Importantly, proteomic studies require comprehensive coverage of proteins contained in the system of interest. Advantages of immobilised pH gradients (IPGs), as opposed to conventional carrier ampholyte gradients, include their stability, reproducibility, and high protein load ability, which is an important attribute for proteomics where proteins are excised from gels for subsequent identification and characterisation.

Proteomic studies also require gels with varying pore size in order to separate and analyse small and large molecules. While commercial gels having relatively small pore size are readily available, the production of gels having large pore sizes continues to have its setbacks. Previous studies have shown that the pores in the gel, as a function of %C_B._S, progressively open up at both very low and very high cross-linker values. The relationship between pore size and %T, by contrast, is quite straightforward and assumes an almost linear decay as the %T is progressively increased. A third way for dramatically enlarging pore sizes would be to produce laterally-aggregated matrices, i.e. polyacrylamides that are forced to produce bundles of fibers, during the gelling process, via addition of preformed polymers in the polymerizing solution.

Not all the above methods for enlarging pore sizes are immune from problems. Highly cross-linked gels are turbid, quite hydrophobic, exude solvent and collapse. Also laterally aggregated gels can become opaque and tend not to be so easily reswellable, due to the tightly packed bundles of fibers. It appears that more dilute gels, down to as low as about 3%T provide an increased pore size however this does not guarantee proper movement of macromolecules.

The present inventors have found that non-uniform reswelling volume of IPG gels, due to the fixed buffering groups, causes improper movement of macromolecules. It is believed that upon reswelling of conventional IPG gels, acidic pH gradients take up more rehydration volume than alkaline gradients, as shown in Figure 4.

Ideally, an IPG gel could be produced having large pore size which is easily and uniformly reswollen.

Summary of the invention

In work leading up to the present invention the inventors have sought to provide a method for improving the protein load and resolution of immobilised pH gradient (IPG) gels. The present invention provides an immobilised pH gradient (IPG) gel that is more uniformly reswollen than currently available gels. Furthermore, the improved IPG gel of the present invention can be produced with larger pore sizes than their conventional equivalents. The combined effects of more uniform reswelling and of diluting the gel matrix favour penetration of large macromolecules and allows for better spot resolution and for the display of a substantially higher number of spots on the IPG gel.

In its broadest aspect, the present invention provides an immobilised pH gradient (IPG) gel comprising an organic acid or organic base, wherein when hydrated, the IPG gel is substantially uniformly swollen along the pH gradient of the gel. The term "hydrated" includes rehydration of a gel from a partially or fully dehydrated state.

Preferably, the organic acid is a carboxylic acid

Preferably, the present invention provides an immobilised pH gradient (IPG) gel comprising (i) polymerised monomeric units of CH₂CRιCONR₂R₃ and cross-linker and (ii) an organic acid or organic base, wherein Ri, R₂ and R are the same or different and are H or optionally substituted alkyl or cycloalkyl, each monomeric unit being the same or different, and wherein when hydrated the IPG gel is substantially uniformly swollen along the pH gradient. Preferably the IPG gel of the present invention is capable of being dehydrated.

In another embodiment the IPG gel of the present invention is capable of being rehydrated.

In a most preferred embodiment, the invention provides an IPG gel comprising

(i) polymerised monomeric units of CH₂CRιCONR₂R₃ and cross-linker and (ii) an organic acid or organic base, wherein Ri, R₂ and R₃ are the same or different and are H or optionally substituted alkyl or cycloalkyl, each monomeric unit being the same or different, and wherein the IPG gel is dehydrated.

Preferably the dehydrated IPG gel is stable and can be stored for a period of time before being rehydrated and used. In one method of preparing an immobilised pH gradient (IPG) gel of the present invention, an organic acid or base is contacted with a polymerised gel. Alternatively, an organic acid or base can be mixed with an IPG gel mixture prior to or during the polymerisation of a gel matrix. In one embodiment, the polymerised IPG gel is washed in water to remove unreacted monomers and/or organic acid or organic base.

Accordingly, in a second aspect the present invention provides a method of

^" preparing an immobilised pH gradient (IPG) gel comprising incorporating an organic acid or organic base into the IPG gel prior to, during or after the polymerisation of the gel, wherein when hydrated the IPG gel is substantially uniformly swollen along the pH gradient.

In one embodiment, the present invention provides a method of preparing an IPG gel, the method comprising contacting a polymerised gel with an organic acid or organic base, wherein when hydrated the IPG gel is substantially uniformly swollen along the pH gradient.

In another embodiment, the present invention provides a method of preparing a dehydrated IPG gel, the method comprising contacting a polymerised IPG gel with an organic acid or organic base, and then dehydrating the polymerised gel.

In a further embodiment, the present invention provides a method of preparing an IPG gel, the method comprising: polymerising a mixture of at least one monomer of formula CH₂CRιCONR₂R₃ and at least one cross-linker to form a gel matrix, wherein Ri, R₂ and R are the same or different and are H or optionally substituted alkyl or cycloalkyl, each monomeric unit being the same or different; and contacting the gel matrix with an organic acid or organic base, such that an amount of the organic acid or organic base is adsorbed in the gel matrix.

Preferably, the method further comprises dehydrating the gel matrix to form a dehydrated IPG gel.

In another embodiment, the present invention provides a method of preparing an IPG gel, the method comprising:

(a) including an organic acid or organic base in a mixture of monomers of CH₂CR]CONR₂R₃ and cross-linker, wherein Ri, R₂ and R₃ are the same or different and are H or optionally substituted alkyl or cycloalkyl, each monomeric unit being the same or different and (b) polymerising the mixture of monomers to form a gel matrix. Preferably, the method further comprises dehydrating the gel matrix after step (b), to form a dehydrated IPG gel.

In a third aspect, the present invention provides an immobilised pH gradient (IPG) gel obtainable by any of the methods of the present invention, wherein when hydrated the IPG gel is uniformly swollen along the pH gradient.

In a fourth aspect, the present invention provides use of an immobilised pH gradient (IPG) gel according to the present invention for separating and/or analysing a mixture of macromolecules.

In a fifth aspect, the present invention provides a kit for analysing or separating macromolecules in a mixture, the kit comprising one or more electrophoresis gel plates according to the first aspect of the invention, buffers and optionally instructions for use.

Brief description of the figures

Fig. 1 is a photograph of two pH 3-10 IPG gels, the one to the left subjected to the usual washing in plain water, the one to the right, instead, equilibrated for 30 min in 100 mM citric acid, followed by two rinses in distilled water.

Fig. 2 is a photograph of two 2-D maps of human platelets run in soft (T% < 4) IPG strips, rinsed in plain distilled water (left panel) or in 100 mM citric acid (right panel).

Fig. 3 is a photograph of 2-D maps of human platelets run in commercial IPG strips (left panel) or in soft IPG gel (T% < 4) strips, rinsed in 100 mM citric acid (right panel).

Fig. 4 is a graph of IPG reswelling versus pH gradient, wherein "dry" commercial IPG gels were measured (cast volume), and then rehydrated and measured again (maximum rehydration volume) .

Detailed description of the invention

In its broadest aspect, the present invention provides an improved IPG gel comprising an organic acid or organic base, wherein when hydrated the IPG gel is substantially uniformly swollen along the pH gradient.

Preferably, the organic acid is a carboxylic acid.

Preferably the carboxylic acid is a polycarboxylic acid. The polycarboxylic acid may be of the formula R(COOH)_n , where R is branched or unbranched optionally substituted alkyl, branched or unbranched optionally substituted alkenyl group, or branched or unbranched optionally substituted alkynyl group and n is an integer from 1 to 4.

Preferably R is C₂ to C₅ alkyl.

In one embodiment a substituent is -OH.

A particularly preferred organic acid is one of formula:

where Ri, R₂ are independently selected from Ci to C₃ alkyl; R₃ is selected from OH, or R₄OH, where R₄ is Ci to C₃ alkyl; and X is absent or is lower alkyl. A particularly preferred organic acid is citric acid.

In one embodiment the organic base comprises an amine group. Alternate basic groups are not excluded.

The term "alkyl group" as used herein refers to a saturated aliphatic hydrocarbon radical including straight-chain, branched chain, cyclic groups, and combinations thereof. Typical alkyl groups and substituted alkyl groups include but are not limited to methyl, ethyl, 1-propyl, isopropyl, 1 -butyl, 2-butyl, tert-butyl, pentyl, isopentyl, hexyl, isohexyl, heptyl and octyl.

The term "alkenyl group" as used herein refers to an unsaturated aliphatic hydrocarbon radical including straight, branched chain, cyclic groups, and combinations thereof, having at least one double bond, of either E or Z stereochemistry where applicable. Examples of alkenyl include but are not limited to ethenyl, 1- propenyl, 2-propenyl, 2-methyl-2-propenyl, 1-butenyl, 1,3-butadienyl, hexenyl, pentenyl, heptenyl and octenyl.

The term "alkynyl group" as used herein refers to an unsaturated aliphatic hydrocarbon group including straight, branched chain, cyclic groups and combinations thereof, having at least one triple bond. Examples of alkynyl groups include but are not limited to include ethynyl, 1-propynyl, 1-, and 2-butynyl,and l-methyl-2-butynyl.

The term "substituted" refers to replacing a group with another group, for example, a carbon group can be replaced with a heteroatom or halide or -OH.

The term "heteroatom" as used herein refers to any atom other than carbon or hydrogen and preferably means oxygen, nitrogen or sulphur. The term "halide atom" as used herein refers to fluorine, chlorine, bromine or iodine.

The term "amine group" as used herein refers to the group -NR₅R₆ wherein R₅ and R₆ are individually selected from the group including but not limited to H, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, alkynyl and substituted or unsubstituted aryl groups.

With regard to the amounts of organic acid or organic base in the IPG gel it will be understood by the skilled addressee that this will vary depending on the method of preparing the IPG gel and the composition and concentration of the IPG . gel. In one embodiment, the IPG gel according to the present invention may comprises trace amounts of organic acid or organic base. In one embodiment, the IPG gel comprises from about 1 μM to about 500mM, more preferably about 1 μM - lOOmM . The concentration of organic acid or organic base in the IPG gel is expected to vary over the length of the IPG gel.

According to a method of preparing an IPG gel according to the present invention, a polymerised IPG gel is contacted with an organic acid or organic base in an effective concentration and for a length of time such that an effective amount of organic acid or organic base is adsorbed by the IPG gel.

As used herein the term "contacting" includes for example, washing, spraying, bathing, and immersing.

The concentration of organic acid or organic base contacted with the IPG gel may be from micromolar to molar concentrations. The amount of time that the IPG gel is in contact with the organic acid or organic base is variable, and can be anything from a few seconds to hours. It is understood, therefore that the gel is contacted with an amount of organic acid or organic base for an effective amount of time that is sufficient for an amount of organic acid or organic base to be adsorbed by the gel. Excess organic acid or organic base can be removed, for example, by washing the gel in water.

In one embodiment, the gel is contacted with about lOmM-lM organic acid or organic base. Preferably, the gel is contacted with about 50mM-500mM organic acid or organic base, more preferably about 50mM-300mM organic acid or organic base, more preferably about 75mM-200mM organic acid or organic base, most preferably about 50mM-100mM organic acid or organic base, more preferably about lOOmM.

In one embodiment, the gel is contacted with an amount of organic acid or organic base for about 5 seconds - 5 hours. More preferably, the gel is contacted with an amount of organic acid or organic base for about 1 minute - 3 hours, more preferably about 10 minutes - 1 hour, more preferably about 20 minutes - 45 minutes, more preferably about 30 minutes.

In an alternate aspect, the present invention provides a method of preparing an IPG gel, the method comprising including an organic acid or organic base in a mixture of monomers of CH₂CRιCONR₂R₃ and cross-linker. Again, it will be understood by a skilled address that the effective concentration of organic acid or organic base included in the mixture will vary from micromolar to molar concentrations, such as for example, about 1 μM to about 500mM organic acid or organic base. In one embodiment, the gel can be washed in, for example water, to remove any unreacted monomers, initiators and organic acids or organic bases. In various embodiments of the invention, the IPG gel is hydrated, rehydrated or dehydrated. As used herein the terms "hydrated" and "hydration" refer to the process whereby ions (and other species) in aqueous solutions are solvated by water. According to the present invention it is understood that when used for electrophoresis, IPG gels are used in a hydrated state. According to the invention, when hydrated the IPG gel is substantially uniformly swollen along the pH gradient of the IPG gel. As used herein "substantially uniformly" refers to the gel being substantially evenly swollen, and preferably more evenly swollen compared to currently available IPG gels.

Preferably, the property of being uniform applies to the thickness of the gel. Accordingly, preferably the thickness of a rehydrated IPG gel of the present invention is substantially uniform along the pH gradient of the gel.

The pH gradient of an IPG gel provides an IPG gel having a more alkaline end and a more acidic end. In one embodiment, addition of an organic acid enhances the reswelling properties of the alkaline end of an IPG gel. In fact, as shown in Fig 1, the alkaline end of the hydrated IPG gel can be thicker than the acidic end after organic acid treatment. In Fig 1 (ii), the alkaline end is overswollen compared to the acidic end, wherein the rippled effect is evidence of overswelling.

In one embodiment, the invention comprises an improved hydrated IPG gel comprising an organic acid or organic base, the gel having an alkaline end and an acidic end, wherein the alkaline end has the same or greater thickness as the acidic end.

In a preferred embodiment of the invention, the alkaline end and acidic end have substantially the same thickness.

As used herein the term "dehydrated" refers to gels having the water present during the polymerisation and washing substantially removed. When dehydrated a gel can shrink to a thickness less than or equal to 10% of their, cast thickness. A dehydrated IPG can reswell with a volume equal to it's cast volume. For example, an 11cm IPG (110mm x 3.3mm x 0.5mm) has a cast volume of 181.5 microlitres. If it is properly dehydrated an IPG gel can take up this volume without any left over.

In one embodiment of the invention, the immobilised pH gradient (IPG) gel comprises polymerised monomeric units of CH₂CR₁CONR2R3 wherein Ri, R₂ and R₃ are the same or different and are H or optionally substituted alkyl or cycloalkyl, each monomeric unit being the same or different.

Preferably, the monomeric units are selected from any of acrylamide, Dimethylacrylamide (DMA) and N-Acryloyl amino propanol (AAP). Other monomers according to the formula are not excluded. Mixtures of monomeric units are encompassed by the present invention. The invention is intended to include mixtures of monomers, for example, as described in PCT/AU02/00666 (incorporated herein by reference). PCT/AU02/00666 describes a hybrid IPG gel comprising a mixture of hydrophilic and hydrophobic monomers to produce improved IPG gels. The IPG gels described in PCT/AU02/00666 are more hydrophobic than commercially available gels, preferably amphiphilic and have improved stability. Further, the described gels can preferably be used with strong hydrophobic extraction solutions such as sulfolane, which provides the ability to extract a wider range of proteins and other macromolecules from a sample. Methods for making or casting gels are well known in the art. Conventionally, acrylamide gel matrix compositions are described as %T/%C, wherein T is the total acrylamide and C is the amount of crosslinking agent.

Preferably, the gel matrix comprises about 2.5-10.0% total acrylamide concentration at a cross-link density of 2-15%. Preferably, the gel matrix comprises about 2.5-8% total acrylamide concentration. More preferably, the gel matrix comprises about 2.5-7% total acrylamide concentration, more preferably about 2.5-6% total acrylamide concentration, more preferably about 3-5% total acrylamide concentration. Most preferably, the gel matrix comprises about 4% total acrylamide concentration. In one embodiment, the immobilised pH gradient (IPG) gel comprises a cross- linker. Various cross linkers are known by the skilled addressee, including for example, cross-linkers used in commercial IPGs such as bis-acrylamide, diacroyl piperazine, DATD, N,N'-diallyl-tartardiamide or piperazine di-acrylamide (PDA). Alternatively, an IPG gel can be cross-linked by a reducible cross-linker such as bis-acryloyl cystamine (BAG) (CH₂=CHCONHCH₂CH₂S-)₂, as described in PCT/AU02/00768 (herein incorporated by reference). Other cross-linkers are not excluded. Preferably the crosslinker is 1,4-Bis (acryloyl)piperazine.

Preferably, the cross-link density is about 2-15%. More preferably, the crosslink density is about 3-12%, more preferably about 5-10%,. Most preferably the cross- link density is about 6%.

In a preferred embodiment the gel matrix comprises 3.75%T/6%C polyacrylamide solution. That is, the gel matrix comprises 3.75% total acrylamide of which 6% is from cross-linking 1,4-Bis (acryloyl)piperazine.

The invention is intended to include IPG gels comprising any suitable pH gradient. In one embodiment, the immobilised pH gradient is in the range of about pH 2 to 12. In alternate embodiments the immobilised pH gradient can be selected from a range of pH gradients including, for example about pH 2-10, about pH 3-10, about pH

4-12, about pH 4-10, about pH 5-9, about pH 3-8, and about pH 5-7. Other pH ranges are not excluded.

It is understood that the immobilised pH gradient gel is an IPG gel strip or an

IPG gel slab. Preferably, a gel slab is a whole sheet of gel as polymerised. Preferably, a strip is typically a narrow (between 3 and 10mm wide) piece of a slab cut out and used for 2-D gels. Preferably, a slab is anything wider than what would be typically used for 2-D gels. Typically, commercial IPGs are 3.3mm wide.

Preferably an IPG gel strip is attached to a backing sheet. Preferably, the backing sheet is a plastic backing sheet. Preferably the plastic is treated plastic. Preferably, the treatment causes the polymerising acrylamide to crosslink to the plastic and thus helps to further stabilise the gel. Preferably, IPG gels according to the present invention are tested for stability according to standard methods as described in

1) Kirkwood, T.B.L Predicting the stability of biological standards and products. Biometrics 33:736-742 (1977)

2) Porterfield, R.I, and Capone, J.J. Applications of Kinetic models and Arrhenius methods to product stability evaluations. Med. Devices Diagn. Industry April 1984, pg

45-50.

3) Kennon, L. Use of models in determining chemical pharmaceutical stability. J. Pharm. Sci. 53: 815-818 (1964) which references are incorporated herein by way of reference. Preferably, IPG gels according to the invention are tested for chemical stability to acidic or alkaline conditions.

In further embodiments the present invention provides a kit for analysing or separating macromolecules in a mixture, the kit comprising one or more IPG gels according to the first aspect of the invention, buffers and optionally instructions for use. Preferably, the macromolecule is a proteinaceous macromolecule, preferably in a mixture of proteins.

In a preferred embodiment, the kit further comprises any one or more of the following: urea, thiourea, CHAPS, carrier ampholytes and an electrophoresis apparatus. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Examples of the invention: material and methods

Reagents

Urea, thiourea, 3-[(3-Cholamidopropyl)dimethylammonio]-l-propanesulfonate

(CHAPS), iodoacetamide (IAA), tributylphosphine (TBP), all Immobilines and sodium dodecyl sulfate (SDS) were obtained from Fluka Chemie (Buchs, Switzerland). Tris(hydroxymethyl)aminomethane and DL-dithiothreitol (DTT) were from Sigma, St. Louis, MO, USA. All the Ampholines, bromophenol blue and agarose were from Pharmacia-LKB (Uppsala, Sweden). Acrylamide, N,N'-methylenebisacrylamide and N,N,N'N'-tetramethylethylenediamine (TEMED) were from Bio-Rad Labs (Richmond, CA). Trichloro acetic acid (TCA), Acetone, Chloroform, methanol and citric acid were from Merck (Darmstadt, Germany).

IPG pH 4 - 10 T% 3.75 ; C% 6 : Acrylamide - 1.4-Bis (acryloyl)piperazine

Dimension of IPG is 18 H x 16 L x 0.075 thickness The support is gel bond pag-film

For polymerization: TEMED 6 μl APS 40 % 12 μl Over night room Temperature >

20 min. at 4 °C for facility the separation between IPG and gel casting

Washing soft IPG with citric acid > The soft IPG after polymerization was washed for 30 min. in 100 mM citric acid followed by two rinses (2*10mins) in distilled water to remove excess of citric acid and then in 2% of glycerol (l*10mins). All washing steps were performed with gentle agitation at room temperature. The rate volume of each wash solution and soft IPG gel was calculated as 10:1 (v/v). The volume of wash solution used was 200ml.

The gel was dried at room temperature with a fan or ventilator. Once the gel was dry, it was covered with mylar film and stored at - 20^DC

Platelet preparation Fresh whole blood was collected from normal healthy volunteers. Each blood sample was processed individually and was mixed with a sodium citrate stock solution to a final concentration of 10% v/v of the anticoagulant. Platelets isolation was caπied out as previously described in Gibbins, J., Asselin; J., Famdale, R„ Barnes, M., Law, C. L.,Watson, S. P. J Biol Chem 1996, 271, 18095-18099. Briefly, upon addition of warmed (30°C) acid citrate dextrose (ACD) solution (117 mM sodium citrate, 282 mM glucose, 78 M citric acid) to a concentration of 7% v/v, the blood was centrifuged for 20 min at 200xg at room temperature to obtain platelet rich plasma (PRP). Following removal of plasma, the platelets were washed in Tyrode— HEPES (134 mM NaCl, 0.34 mM Na₂HPO₄, 2.9 mM KCl, 12 mM NaHCO₃, 20 mM HEPES, 5 mM glucose, 1 mM MgCl₂ pH 7.3 and EGTA 1 mM) containing ACD (7%, v/v). The platelet pellet was then resuspended in Tyrodes- HEPES, to a concentration of 2 χl0^s/ml or 1 xlO^/ml and following addition of 20 1 of a protease inhibitor cocktail with immediately frozen in liquid nitrogen prior to storage at -80°C. Pellets of frozen platelets was reduced, alJάlated and delipidated, prior isoelectric focusing .

Reduction and All viation

The protein platelets of the stock preparation (5 μg/μl) were added to a 10% SDS, 3% DTE, 40 M Tris and 0.1 mM EDTA solution and treated for 5 min at 100^QC in a method as escribed .Herbert B. et al., Electrophoresis 2001, 22, 204-57. 5 Preparation of dilipidated platelets for 2-D PAGE

After reduction and alkylation, the sample was dilipidated with a solution consisting of tri-n-burylphosphate:acetone:methanol (1:12:1) and cooled in ice. Fourteen (14) mL of this mixture were added to the SDS-solubilized platelets to a final acetone concentration of 80% and incubated at 4°C for 90 min. The precipitate was pelleted by centrifugation at 2800 g for 20 min at 4°C. After washing the pellet with the same delipidizing solution, it was centrifuged again and then air-dried. The pellet was finally dissolved in the focusing solution, i.e. 8 M urea, 1 M thiourea, 4% w/v CHAPS, 65 mM

DTE, 40 mM Tris and 0.1 mM EDTA.

Rehvdration of IPG strips

Two types of strips were used:

1) commercial IPG strips (18. cm long, 3 mm wide, 0.5 mm thick) in a non-linear pH 3-

10 interval, containing 4% polyacrylamide, and 2) Proteome IPG strips, having the same size and IPG composition as the commercial

IPG strips, but made with variable matrix content, ranging from as low as 3%T up to

3.75% T polyacrylamide.

All strips were reswollen in the same solution, consisting of: 8 M urea, 1 M thiourea, 4% CHAPS, 65 mM DTE and a 1.6% carrier ampholyte cocktail, containing 50% of the pH 3.5-10, 30% of the pH 4-8, 10% of the pH 8-10.5 and 10% of the pH 7-9 intervals. Reswelling of the IPG strips was carried out overnight at 12°C.

Two-dimensional gel electrophoresis 1 Isoelectric focusing (IEF) of proteins in IPG strips (first dimension)

All samples were routinely loaded, onto caps at the cathodic end of the rehydrated IPG strips, and covered with low-viscosity paraffin oil. IEF was run at 18°C. The voltage was progressively increased from 300 to 3000 N during the first 3 hrs, followed by 5000 N for a total of 100 kV. Before the 2^nd dimension run, the IPG strips were equilibrated for 15 min with a solution of 40 mM Tris-acetate buffer, pH 6.8 containing 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS and 2.6% (w/v) DTE.

2 Separation of proteins in SDS-PAGE (second dimension)

In the second dimension, proteins were separated based on their size in 8-18%T gradient polyacrylamide gels having the following dimensions: 180 x 160 x l.5 mm.

The electrophoretic equipment included a Protean II Multi-Cell vertical chamber and a power supply 3000x1 (Bio-Rad, Hercules, CA, USA). No stacking gel was employed. The IPG strips were embedded with 0.5 % (w/v) melted agarose prior to the run on the SDS-PAGE slabs. The agarose contained 0.001 % (w/v) bromophenol blue as a tracking dye. The gels were run at 45 mA/gel constant current and maintained at a temperature between 8 and 12°C.

Gel staining

After separation in SDS-PAGE gels, the proteins were visualized by a double staining procedure: first the methyl-trichloroacetate negative staining of Candiano, G., Porotto, M., Lanciotti, M., Ghiggeri, G.M., Anal. Biochem. 1996, 243, 245-250, followed by the silver staining of Oakley, B.R., Kirsch, D.R., Morris, N.R., Anal. Biochem. 1980, 105, 361-366.

Results Fig. 1 compares two pH 3-10 IPG gels, the one to the left subjected to the usual washings in plain water, the one to the right, instead, equilibrated for 30 min in 100 mM citric acid, followed by two rinses in distilled water. It can be noticed that the latter one, in the alkaline region, shows the typical ripples of a slight overswelling, as though it were more charged than the acidic counter-part. Upon drying (followed by storage, if necessary) and reswelling in the sample solution, in preparation for two-dimensional mapping, the IPG strips washed in citric acid exhibited a much better tendency to reswell and become impregnated with sample solution. The strips treated only with distilled water showed a thinner alkaline region and a considerably lower tendency to properly reswell in the sample solution. The results of this differential treatment can be appreciated in Fig. 2, which displays 2-D maps of purified human platelets. Two major improvements can be appreciated in the gels which had undergone citric acid washing: the alkaline gel region in richer in spots; overall, more spots appears throughout the entire gradient and high M_r macromolecules (>200 kDa) are revealed as much more intense spots.

Discussion on the mechanism of organic acid washing

It has been reported that the basic gel region of IPG gels has a reduced swelling capability as compared to its acidic counterpart. In light of the present invention, however, there seems to be a peculiar effect of washing the IPG gel in organic acid, namely the superior reswelling ability of the IPG gel in the alkaline gel region. It appears as though, after the rinsing of the excess organic acid in distilled water, a gradient of this tri-carboxylic acid remains trapped into the IPG matrix, from almost nill at the acidic gel region to substantially higher amounts in its basic counterpart. This gradient helps in obtaining a uniform reswelling of the IPG strip, since carboxyl groups are more heavily hydrated than amino groups. The combined effects of uniform reswelling and of diluting the gel matrix favour penetration of large macromolecules (>200 kDa) and allows for better spot resolution and for the display of a substantially higher number of spots also in the 30-60,000 Da region. A delipidation step in tri-n-butylphosphate:acetone:methanol (1:12:1) appears also to substantially improve spot focusing and greatly diminish streaking and smearing of spots in all regions of the pH gradient.

The inventors have noted that when applying a voltage gradient to a reswollen IPG strip, during the initial phases of the focusing process, a stronger refractive index boundary moves away from the cathode, bound "to the opposite electrode. This ionic boundary could be the excess organic acid being depleted by the applied electric field gradient. The combined benefits of an even gel reswelling all along the pH gradient strip, combined with the higher porosity of the dilute matrix, is suggested to be beneficial on both fronts, in helping larger macromolecules to penetrate within the gel pores and in minimizing protein-matrix interactions. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

1. An immobilised pH gradient (IPG) gel comprising an organic acid or organic base, wherein when hydrated, the IPG gel is substantially uniformly swollen along the pH gradient of the gel.

2. An immobilised pH gradient (IPG) gel comprising (i) polymerised monomeric units of CH₂CRιCONR₂R₃ and cross-linker and (ii) an organic acid or organic base, wherein Ri, R₂ and R₃ are the same or different and are H or optionally substituted alkyl or cycloalkyl, each monomeric unit being the same or different.

3. The immobilised pH gradient (IPG) gel according to claims 1 or 2, wherein the IPG gel is dehydrated.

4. The immobilised pH gradient (IPG) gel according to claims 1 or 2, wherein the IPG gel is hydrated.

5. The immobilised pH gradient (IPG) gel according to claim 5, wherein the IPG gel is rehydrated.

6. The immobilised pH gradient (IPG) gel according to claim 2, wherein the hydrated IPG gel has an acidic end and an alkaline end and the alkaline end has substantially the same or greater thickness as the acidic end.

7. The immobilised pH gradient (IPG) gel according to claim 2, wherein the hydrated IPG gel has an acidic end and an alkaline end and the alkaline end has substantially the same thickness as the acidic end.

8. The immobilised pH gradient (IPG) gel according to any one of claims 1 to 7, wherein the organic acid is a carboxylic acid.

9. The immobiiised pH gradient (IPG) gel according to claim 8, wherein the carboxylic acid is a polycarboxylic acid.

10. The immobilised pH gradient (IPG) gel according to claim 9, wherein the polycarboxylic acid comprises the formula R(COOH)_n , where R is branched or unbranched optionally substituted alkyl, branched or unbranched optionally substituted alkenyl group, or branched or unbranched optionally substituted alkynyl group and n is an integer from 1 to 4.

11. The immobilised pH gradient (IPG) gel according to claim 10, wherein R is C₂ to C₅ alkyl.

12. The immobilised pH gradient (IPG) gel according to any one of claims 1 to 7, wherein the organic acid comprises a formula :

where Ri, R₂ are independently selected from to C₃ alkyl;

R₃ is selected from OH, or R₄OH, where R₄ is Ci to C alkyl; and X is absent or is a lower alkyl.

13. The immobilised pH gradient (IPG) gel according to claim 12, wherein the organic acid is citric acid.

14. The immobilised pH gradient (IPG) gel according to any one of claims 1 to 7, wherein the organic base comprises an amine group.

15. The immobilised pH gradient (IPG) gel according to any one of claims 1 to 14, wherein the IPG gel comprises from lμM to lOOmM organic acid or organic base.

16. The immobilised pH gradient (IPG) gel according to any one of claims 2 to 15, wherein the monomeric units of CH₂CRιCONR₂R₃ are selected from the group consisting of: acrylamide, Dimethylacrylamide (DMA) and N-Acryloyl amino propanol (AAP), or a mixture thereof.

17. The immobilised pH gradient (IPG) gel according to any one of claims 2 to 16, wherein the cross-linker is selected from the group consisting of bis-acrylamide, 1,4- Bis(acyloyl) piperazine, piperazine di-acrylamide (PDA), and bis-acryloyl cystamine (BAG) (CH₂=CHCONHCH₂CH₂S-)₂, or a mixture thereof

18. The immobilised pH gradient (IPG) gel according to any one of claims 1 to 17, comprising a pH gradient of about pH 3-10.

19. The immobilised pH gradient (IPG) gel according to any one of claims 1 to 17, comprising a pH gradient of about pH 4-12.

20. A method of preparing an immobilised pH gradient (IPG) gel comprising incorporating an organic acid or organic base into the IPG gel prior to, during or after the polymerisation of the gel, wherein when hydrated the IPG gel is substantially uniformly swollen along the pH gradient.

21. A method of preparing an IPG gel, the method comprising contacting a polymerised gel with an organic acid or organic base, wherein when hydrated the IPG gel is substantially uniformly swollen along the pH gradient.

22. A method of preparing a dehydrated IPG gel, the method comprising contacting a polymerised gel with an organic acid or organic base, and then dehydrating the polymerised gel.

23. A method of preparing an IPG gel, the method comprising: polymerising a mixture of at least one monomer of formula CH₂CRιCONR₂R₃ and at least one cross-linker to form a gel matrix, wherein Ri, R₂ and R₃ are the same or different and are H or optionally substituted alkyl or cycloalkyl, each monomeric unit being the same or different; and contacting the polymerised gel matrix with an organic acid or organic base, such that an amount of the organic acid or organic base is adsorbed in the gel matrix.

24. The method of preparing an IPG gel according to claim 23, wherein the gel matrix is contacted with about lOmM-lM organic acid.

25. The method of preparing an IPG gel according to claim 23, wherein the gel matrix is contacted with about 50mM-500mM organic acid.

26. The method of preparing an IPG gel according to claim 23, wherein the gel matrix is contacted with about 50mM-100mM organic acid.

27. The method of preparing an IPG gel according to claim 23, further comprising the step of washing the gel matrix to remove excess organic acid or organic base.

28. The method of preparing an IPG gel according to any one of claims 23-27, further comprising the step of dehydrating the gel matrix to form a dehydrated IPG gel.

29. A method of preparing an IPG gel, the method comprising:

(a) including an organic acid or organic base in a mixture of monomers of CH₂CRιCONR₂R₃ and cross-linker, wherein Ri, R₂ and R₃ are the same or different and are H or optionally substituted alkyl or cycloalkyl, each monomeric unit being the same or different and (b) polymerising the mixture of monomers to form a gel matrix.

30. The method of preparing an IPG gel according to claim 29, the method further comprising dehydrating the gel matrix after step (b), to form a dehydrated IPG gel.

31. The method of preparing an IPG gel according to any one of claims 20-30, wherein the organic acid is a polycarboxylic acid.

32. The method according to claim 31, wherein the polycarboxylic acid is citric acid.

33. An immobilised pH gradient (IPG) gel obtainable by the method of any one of claims 20-32, wherein when hydrated the IPG gel is uniformly swollen along the pH gradient.

34. Use of an immobilised pH gradient (IPG) gel according to any one of claims 1- 19 for separating and/or analysing a mixture of macromolecules.

35. A kit comprising an immobilised pH gradient (IPG) gel according to any one of claims 1-19 or 33, a buffer, and optionally instructions for use.

36. The kit according to claim 35, further comprising any one or more of the following: urea, thiourea, CHAPS, carrier ampholytes and electrophoresis apparatus.