2-D电泳中样品制备

生物秀-专心做生物生物秀论坛—学术交流、资源共享与互助社区http:// 生物部落—生物医药人网络家园http:// 生物百科—生命科学第一百科http:// 生物秀知道—生命科学问题解决之道！http:// 易生物-领先的生物医药商务平台ReviewMargaret M.Shaw 1,2Beat M.Riederer 1,21Institut de Biologie Cellulaireet de Morphologie,Universitéde Lausanne,Lausanne,Switzerland 2Centre de Neurosciences Psychiatriques,Hôpital de CERY ,Prilly,SwitzerlandSample preparation for two-dimensional gel electrophoresisThe choice of sample preparation protocol is a critical influential factor for isoelectric focusing which in turn affects the two-dimensional gel result in terms of quality and protein species distribution.The optimal protocol varies depending on the nature of the sample for analysis and the properties of the constituent protein species (hydro-phobicity,tendency to form aggregates,copy number)intended for resolution.This review explains the standard sample buffer constituents and illustrates a series of pro-tocols for processing diverse samples for two-dimensional gel electrophoresis,includ-ing hydrophobic membrane proteins.Current methods for concentrating lower abun-dance proteins,by removal of high abundance proteins,are also outlined.Finally,since protein staining is becoming increasingly incorporated into the sample preparation procedure,we describe the principles and applications of current (and future)pre-elec-trophoretic labelling methods.Keywords:Detergents /Prefractionation /Prelabelling /Protease inhibitors /Review /Thiourea /Two-dimensional gel electrophoresis PRO 0471Contents1Introduction ..........................14082Sample buffer constitutents .............14092.1Urea ................................14092.2Thiourea .............................14092.3Detergents ...........................14102.4Reducing agents ......................14102.5Carrier amphloytes/Immobilines ..........14112.6Protease inhibitors .....................14113Recent advances in sample preparation methodology .........................14123.1Sample prefractionation ................14123.2Resolution of “elusive”proteins ..........14143.3Prelabelling applications ................14154Conclusions .. (14165)References ...........................14161IntroductionTwo-dimensional gel electrophoresis (2-DE)is a protein separation technique that combines two different electro-phoretic methods,that is gel IEF in the first dimension (in which proteins are separated according to p I )and SDS-PAGE in the second dimension (separation according to molecular weight (M r )).The aim of sample preparation for 2-DE is to convert the native sample into a suitable physicochemical state for first dimension IEF while preserving the native charge and M r of the constituent proteins.In many cases this means that the proteins of the sample need to be solubi-lised,disaggregated,denatured and reduced.However,the specific sample preparation method depends first and foremost on the aim of the separation.For example,if the aim of the first dimension is to resolve the isoelectric points of soluble aggregates,then the IEF needs to be carried out under nondenaturing,nonreducing conditions and the aim of the sample preparation method would be to achieve protein solubilisation without disaggregation.In many cases solubilisation under these conditions is incomplete and only the supernatant from the centrifuged sample can be applied to the first dimension.In nondena-turing,nonreducing IEF partial solubilisation of thylakoid membrane proteins has been achieved with the nonionicCorrespondence:Dr.B.M.Riederer,IBCM,Rue du Bugnon 9,1005Lausanne,SwitzerlandE-mail:beatmichel.riederer@ibcm.unil.ch Fax:141-21-692-5105Abbreviations:ASB14,myristic amidosulphobetaine;SB3-10,caprylyl sulphobetaine;TBP ,tributyl phosphine;TCEP ,Tris (2-carboxyethyl)phosphine1408Proteomics 2003,3,1408–1417DOI 10.1002/pmic.200300471 2003WILEY-VCH Verlag GmbH &Co.KGaA,WeinheimProteomics 2003,3,1408–1417Sample preparation 1409detergents Triton X-100and N -dodecyl-b -D -maltoside [1].Triton X-100and dodecyl maltoside are considered non-denaturing as they are more efficient at breaking lipid-lipid and lipid-protein interactions than protein-protein interac-tions.Probably the aim of most 2-D gel applications is,however,to resolve as many proteins as possible within a particular p I /M r range,to facilitate comparison of two sets of data samples.In this case,it is preferable to carry out IEF under denaturing and reducing conditions.The following gives an account of the individual sample buffer constitu-ents,their function,possible problems associated with each buffer component and recent advances or modifica-tions which improve the final 2-D gel outcome and facil-itate representation of a certain species of proteins which,due to their physicochemical properties,have until recently,remained unresolved on the 2-D gel map.2Sample buffer constitutents2.1UreaSample solutions for first dimension separations under denaturing conditions always include urea.This neutral chaotrope denatures proteins by disrupting noncovalent and ionic bonds between amino acid residues.Its neutral charge renders it ideal for IEF as it remains in the gel dur-ing focusing and does not migrate.It may be included in the sample solution at a concentration of ,5M to as high as 9.8M .However,urea solutions are not stable.Sponta-neous degradation of urea to cyanate (Fig.1)occurs at room temperature.If left overnight at roomtemperatureFigure 1.Cyanate formation and carbamylation.10M urea will degrade to equilibrium to form 20m M cyanate.Reaction of cyanate ions with the amine groups on proteins (carbamylation)removes the positive charge on the amine,which affects the IEF result.Car-bamylation is not limited to amine groups.Lippincott and Apostol [2]investigated carbamylation of sulphhy-dryl groups on cysteine residues and found that carba-mylation of cysteines readily occurs under slightly acidic conditions (pH 6)but that carbamylmercaptans are unstable in an alkaline environment.The issue of carba-mylation presents a potential problem when IPG strips are rehydrated overnight with the prepared sample in strip rehydration solution.However,the overall quality of the 2-D gel map only appears to be affected at extended strip rehydration times of around 24h [3].To reduce the effects of carbamylation the cyanate scaven-ger,spermine,can be added to the sample solution or strip reswelling solution [4].2.2ThioureaUse of thiourea in combination with urea for IEF was first reported by Thierry Rabilloud in 1996at the Siena 2-D electrophoresis conference [5].As a result a whole vari-ety of proteins previously elusive to the 2-D gel map could be resolved [6–8].Thiourea has particularly been shown to improve solubilisation of hydrophobic mem-brane proteins [9,10].One disadvantage of thiourea,reported by Galvani et al.[11],is that at pH values of 8.5–9,which coincide with the pH of the buffer for equili-bration after the first dimension (pH 8.8),the sulphur atom of thiourea is as reactive as the SH group of cys-teine.As a consequence thiourea present in the first di-mension gel scavenges the iodoacetamide during equili-bration after the first dimension resulting in poor alkyla-tion of proteins.To counteract this,Galvani et al.[11]recommend that thiourea should first be omitted from the solubilising solution and incorporated after the sam-ple has been reduced and alkylated,prior to first dimen-sion IEF .Reduction and alkylation at the sample prepa-ration stage has the additional advantage of inhibiting carbamylation since (i)DTT acts as an effective scaven-ger of cyanate,and (ii)alkylation prevents further reac-tion with cyanate ions [2].Thiourea is generally used at concentrations of 2M in conjunction with 5–7M urea al-though Musante et al.[7]reported a reduction in protein resolution associated with increasing concentrations of thiourea and consequently limited the concentration of thiourea to 0.5M .The loss in protein resolution may pos-sibly be a result of poor transfer to the second dimen-sion,as thiourea additionally tends to inhibit SDS-protein binding,or of improved solubilisation of lipids,which affect resolution on 2-D gels.2003WILEY-VCH Verlag GmbH &Co.KGaA,Weinheim1410M.M.Shaw and B.M.Riederer Proteomics2003,3,1408–14172.3DetergentsTraditionally,sample buffer protocols for2-DE included the anionic detergent SDS[12].Since protein-detergent micelles formed on SDS treatment carry an overall nega-tive charge,inclusion of SDS in sample buffers for IEF seems illogical.However,SDS was found not to interfere in IEF if used together with an excess of nonionic or zwit-terionic detergent(e.g.NP-40,Triton X-100,CHAPS)and it was suggested by O’Farrell[12](who used NP-40in conjunction with SDS)that the nonionic detergent forms mixed micelles with the SDS which migrate to the anode. Ames and Nikaido[13]investigated the criteria for SDS compatibility and established that the NP-40:SDS con-centration ratio should be at least8:1in order to avoid a streaking effect.In addition the final concentration of SDS in the IEF sample buffer should be below0.25%[14].Some inconveniences of using SDS for protein solubilisa-tion for2-DE application are discussed in a review by Mol-loy[5]who also mentions that the electrophoretic removal of SDS from the proteins during focusing may in some cases result in in-gel isoelectric precipitation.Indeed, even small amounts of SDS have been found to remove all the detergent from the gel due to the formation and an-odic migration of mixed micelles leaving the proteins exposed to an environment containing little or no deter-gent[15].An additional factor concerning SDS solubilisa-tion/denaturation is that SDS is not suitable for all appli-cations,since strongly acidic proteins do not bind SDS. The alternative for such proteins would be to use cationic detergents,which are,however,not widely investigated in 2-DE applications.Nonionic detergents are favoured for nondenaturing IEF as most,such as Tween80,NP-40and Triton X-100are mild detergents which retain enzyme activities.The origi-nal protocols of O’Farrell[12]and Klose[16]utilised NP-40and Triton X-100.These detergents are generally used at concentrations between0.4–4%,although as high as 10%NP-40has been used in sample buffers by Lenstra and Bloemendal[17].N-dodecyl-b-D-maltoside is also a nonionic detergent which has been used at a final con-centration of0.47%[1].The zwitterionic detergent,CHAPS has since been demonstrated to be more effective at solubilisation[18]. CHAPS is currently the most commonly used detergent in standard procedure for2-DE and belongs to the class of linear sulphobetaine surfactants which also include caprylyl sulphobetaine(SB3–10),and amidosulphobe-taine14(ASB14).Urea tolerance is a factor which must be taken into account when selecting detergents for IEF sample buf-fers.Some of the powerful detergents disadvantageously form inclusion compounds with urea,for example,SB3–10is a powerful surfactant which is however,compatible only with low concentrations of urea,whereas the shorter-tailed,less efficient detergent,SB3–8,is compatible with high concentrations of urea[19].ASB14is,however,an efficient surfactant compatible with9M urea.Structures of several nonionic and zwitterionic detergents are shown in Fig.2.2.4Reducing agentsReduction of disulphide bonds aids solubilisation of com-plex mixtures of proteins.This is commonly achieved with free thiol-containing reducing agents such as dithiothrei-tol(DTT),dithioerythritol(DTE)or2-mercaptoethanol[20]. However,2-mercaptoethanol is no longer widely used in carrier ampholyte IEF,particularly where basic proteins are of interest,since it ionises at the alkaline end of the gel and is driven electrophoretically along the pH gradi-ent,apparently sweeping away focused carrier ampho-lytes,resulting in a disturbance in this region[21].In addi-tion,DTT,DTE and an alternative reducing agent,tris(2-carboxyethyl)phosphine(TCEP),which are also charged at alkaline pH,migrate towards the anode,depleting the basic end of the gel during IEF,which results in re-oxida-tion of reduced S-S bonds[22].In the absence of an alkyl-ation step during sample preparation this results in pre-cipitation of some proteins(particularly keratins and kera-tin-associated proteins from hair and wool which are rich in disulphide bonds)and often in spurious spots in the alkaline pH region due to formation of“scrambled”disul-Figure2.Nonionic and zwitterionic detergents.2003WILEY-VCH Verlag GmbH&Co.KGaA,WeinheimProteomics2003,3,1408–1417Sample preparation1411phide bridges[23].The noncharged reducing agent tribu-tyl phosphine(TBP)overcomes this problem.DTT and DTE are both used in the concentration range of20–100m M,whereas higher concentrations are required for the less potent2-mercaptoethanol.TBP has been used at a concentration of2m M[24].2.5Carrier ampholytes/ImmobilinesThe addition of carrier ampholytes to the solubilising buf-fer has several advantages.First,where IPG strips are used,carrier ampholytes are useful in inhibiting interac-tions between hydrophobic proteins and Immobilines which tend to occur at the basic end of the gel,leading to a streaking effect due to precipitation[25,26].Carrier ampholytes additionally scavenge cyanate ions and help in the precipitation of nucleic acids during centrifugation. Carrier ampholytes are generally used at a concentration of0.5–2%,although2%is normally only recommended if protein solubilisation is a problem.Table1provides a list of different sample buffers used for solubilising various specimens for2-DE.2.6Protease inhibitorsThe denaturing properties of most sample buffers are often sufficient to inhibit the action of proteases[3,20]. However,cathepsin C,several carboxypeptidases and endoproteinases,chymotrypsin,trypsin,plasmin and proteinase K are examples of some proteases which remain active in1mg/mL SDS.Thus,depending on the sample buffer employed,inclusion of protease inhibitors may be necessary.In addition,protease inhibitors are useful if lengthy sample manipulations are carried out prior to the first dimension loading.This is exemplified by the work of Olivieri et al.[33]who observed that failure to add protease inhibitors during the lengthy procedure of erythrocyte membrane preparation,which is carried out in physiological buffers providing favourable conditions for protease activity,led to massive degradation of high molecular weight proteins,resulting in a mixture of low molecular mass(,50kDa)proteins and peptides on the 2-D gel map.Some commercially available protease in-hibitors and their target proteases are summarised in Table2.Table1.Examples of solubilisation buffers which have been used for preparation of different samples for2-DE.Sample Urea Thiourea Reducingagent Detergent CarrierampholytesReferenceChinese Hamster Ovary cells (exogenously expressing membrane proteins)7M2M50m MDTT2m M TCEP-HCl2%C80or2%CHAPSor2%ASB140.5%(3–10)Henningsen etal.[27]Mouse brain5M2M60m MDTT 2%CHAPS2%(3–10)Riederer andShaw[3]Bacterial outer membrane proteins(after carbonatewashing)7M2M30m MDTT1%ASB140.5%TritonX-1000.5%(3–10)Molloy et al.[28]E.coli outer membrane proteins (after carbonate washing)7M2M2m MTBP1%ASB140.5%(3–10)Molloy et al.[29]Human plasma8M None10m MDTE 2%CHAPS0.8%(4–8)Liberatori et al.[30]Mouse liver8M None60m MDTT 0.5%TritonX-1002%(3–10)O’Connell andStults[31]Disaggregated human kidney tissue 9M None65m MDTE4%CHAPS4%(9–11)Sarto et al.[32]2003WILEY-VCH Verlag GmbH&Co.KGaA,Weinheim1412M.M.Shaw and B.M.Riederer Proteomics2003,3,1408–1417Table2.Protease inhibitors and their targetsProtease inhibitor Target Recommendedworkingconcentration APMSF Plasma serineproteases10–20m M Aprotinin Serine proteases0.01–0.3m M Bestatin Aminopeptidases40m g/mL Dichloroisocoumarin Serine proteases1–43m g/mL Disodium EDTA Metalloproteases100m ME-64Thiol proteases 1.4–2.8m M Leupeptin Serine and thiolproteases1m M Pepstatin Acidic proteases1m MPMSF Serine proteases100–1000m M Phosphoramidon ThermolysinCollagenaseMetalloendoproteases7–569m MTLCK.HCl TrypsinThiol proteases37–50m g/mLTPCK ChymotrypsinThiol proteases70–100m g/mL3Recent advances in sample preparation methodologyThere are three areas in sample preparation methodology which have been the subject of significant advance in recent years.These are(1)sample prefractionation,(2) resolution of“elusive”proteins,and(3)prelabelling appli-cations.3.1Sample prefractionationSeveral examples can be provided of comparative2-D gel studies in vivo where little or no differences have been observed in the2-D gel maps between the control and test groups(Riederer and Shaw,unpublished data; Hondermarck et al.[34]).In many cases,even after some anatomical prefractionation(for example,removal of hip-pocampus or prefrontal cortex from whole brain)such studies fail to show differences due to the heterogeneity of cell types within a specimen:a protein marginally up-regulated in one cell type by a particular drug treatment may be missed due to the majority of other unaffected cell types in the same sample.Similarly,some proteins in the sample may comigrate to the same spot,clouding the result.This has often led researchers to abandon the in vivo study in favour of a homogenous cell line.However, examples of enrichment of cells according to specific type do exist:fluorescence-activated cell sorting(FACS) of antibody-bound cells enables the collection of specific cell types from clinical specimens.One unique example which does not require antibody binding is the isolation of rat b-cells which can be distinguished from other cell types by their autofluorescence.After trypsin digestion of islets,rat b-cells can be FACS-sorted to.95%purity. Some cell types,for example,fibroblasts,neurons or glial cells can be“enriched”from clinical specimens by pro-ducing primary cultures under conditions which select out the cell type of interest and inhibit growth of other cell types.However,these manipulations are likely to result in activation of stress and other signalling pathways which may induce a considerable change in the protein profile of the cell.Subcellular fractionation either from a homogeneous cell type or from tissues composed of heterogeneous cell types provides an additional method of protein exclusion. Classical subcellular fractionation procedures were based mainly on centrifugation techniques[35–37].How-ever,recently developed methods rely more and more on differential resistance or susceptibility of cell components to various extraction buffers,for example the resistance of actin filaments,intermediate filaments and nuclei against nonionic detergents,the resistance of most inter-mediate-sized filament proteins,vimentin and nuclear proteins against the weakly ionic detergent sodium deoxy-cholate(in contrast to actin filaments which are sus-ceptible to solubilisation by this detergent)and the resis-tance of intermediate-sized filaments against nonionic detergents and1.5M KCl[17].Similarly,Molloy et al.[9] were able to partition Escherichia coli membrane proteins from other proteins based on the limited solubility of membrane proteins in solutions conventionally used for IEF(8M urea,4%CHAPS,100m M DTT,40m M Tris base, 0.5%v/v Pharmalyte3–10,150U endonuclease,pH9.5) and their partial solubility in a lysis buffer consisting of5M urea,2M thiourea,2m M TBP,2%w/v SB3–10,2%w/v CHAPS,40m M Tris base,0.5%v/v Pharmalyte3–10, 150U endonuclease,pH9.5.By a similar technique Andréet al.[38]were able to purify intermediate filaments from eukaryotic cells.Using several independent chemical extraction methods, Lenstra and Bloemendal[17]were able to characterise seven distinct subcellular fractions from cultured hamster lens cells on2-D gels which represented the total detect-able protein population of these cells(water soluble pro-teins,membrane proteins,actin filaments,intermediate-sized filaments,microtubular proteins,polyribosomes and nuclei).For2-D gel analysis,these fractions were dis-solved in SDS-containing lysis buffer[39]and,despite the recommendations of Wilson et al.[40],boiled for3min,2003WILEY-VCH Verlag GmbH&Co.KGaA,WeinheimProteomics2003,3,1408–1417Sample preparation1413then brought to9M urea10%NP-40,5%v/v Ampholines pH3–10and5%2-mercaptoethanol.The sum of the2-D gel profiles of these fractions almost completely comple-mented the2-D gel map of total cell lysates prepared by freeze-thawing in MgCl2(5m M),KCl(25m M),50m M Tris HCl pH7.4,treated with DNase I for15min at room tem-perature and diluting with SDS lysis buffer[39].In many cases abundant proteins present a problem on 2-D gel maps as they severely limit the amount of non-abundant protein which can be loaded onto the first di-mension and in addition,the enlarged spots which they produce on the gel may cloud or displace other spots, resulting in inaccurate p I/M r representations of particular proteins or failure to detect proteins which may be signif-icant.An example of a highly abundant protein in serum is albu-min,which may constitute50%of proteins present in serum.The product ProtoClear for albumin removal[41] comprised an affinity column with a monoclonal antibody to albumin,allowing better representation of proteins other than albumin on2-DE gels.However,this product is no longer available.Albumin removal columns are,how-ever,offered by several companies,but these do not ex-clude some nonspecific binding.Fibrinogen is also a highly abundant protein in plasma,which can be removed by affinity chromatography.PlasmaSelect(Martinsried,Ger-many)has developed a fibrinogen adsorption system (“Rheosorb”)consisting of a fibrinogen-specific penta-peptide ligand which selectively removes fibrinogen and fibrin from plasma[42].The basis of this ligand is currently used by Geneva Proteomics(GeneProt,Geneva,Switzer-land)for fibrinogen removal prior to2-DE.In addition to columns for removal of high abundance pro-teins,there also exist commercially available kits for removal of substances which might otherwise interfere in IEF(e.g.PlusOne2-DE clean up kit,Amersham Bio-sciences,Little Chalfont,Bucks,UK).Interfering sub-stances include salts,nucleic acids,lipids and polysac-charides.In some instances salts may induce protein modification[43].Generally,though,salts delay the onset of protein focusing which cannot occur until the ions have migrated to the electrodes.In addition salts increase the conductivity of the IEF gel,as the current increases since resistance is a constant,focusing occurs at a lower volt-age,prolonging the time required for IEF.High salt con-centrations may also cause electroendosmosis(EEO) resulting in uneven water distribution in the gel which forms zones of dehydration and overhydration.The salt concentration for IPG strips when sample is applied by in-gel rehydration should be lower than10m M.Applica-tion via sample cups permits higher salt concentrations (up to50m M).However,proteins may precipitate at the sample application site as they move into the lower salt environment of the first dimension gel[43].Salts can be removed from samples by dialysis,gel filtration,TCA precipitation or use of protein concentration devices(e.g. Centricon columns,Millipore,Walford,UK).Urine and sweat are examples of clinical samples with a high salt content.Urine sample preparation and solubilisation for 2-DE have been described by Anderson et al.[44]and Edwards et al.[45].Lipids bind proteins via hydrophobic interactions,affect-ing their charge and M r.In many cases the protein lipid complex is insoluble in aqueous solution,resulting in fail-ure to enter the first dimension gel.This interaction can be outcompeted by the addition of excess detergent.In the case of lipid-rich tissues such as brain or adipose tissue, excess lipids can be removed by acetone precipitation.Polysaccharides can interfere in IEF by obstructing gel pores.Ultracentrifugation removes high M r polysaccha-rides.Lower M r polysaccharides may be removed by pre-cipitation in TCA,ammonium sulphate or phenol/ammo-nium acetate.Nucleic acid rich samples usually constitute rapidly divid-ing cells,cultured cells or biopsies from fast-growing tumours.Nucleic acids can bind proteins through electro-static interactions,preventing focusing.High M r nucleic acids can additionally clog the pores of the acryl-amide matrix.They can be removed by treatment with nucleases,addition of carrier ampholytes with subse-quent ultracentrifugation[12]or by precipitation with a basic polyamine at high pH[46].In addition to the removal of abundant proteins such as albumin,column chromatography is also useful for the removal of certain contaminants.Desalting columns, for example,function by size exclusion.In addition to size exclusion chromatography,several other chromato-graphic techniques(hydrophobic interaction and re-versed-phase chromatography,ion-exchange and affinity chromatography)[47]can be applied to sample prefrac-tionation for2-DE.However,the chromatographic princi-ple employed depends on the aim of the electrophoretic separation.If only one or a group of proteins are to be studied by 2-DE,enrichment may be carried out by chromatographic methods.However,one technique which enables enrich-ment of many different proteins within a particular p I range is preparative IEF.This is useful if proteins are to be resolved afterwards on high resolution narrow range, first dimension gels,due to the limit to the overall quantity of protein which can be applied to the first dimension and avoids wasteful application of proteins which are outside of the p I range.2003WILEY-VCH Verlag GmbH&Co.KGaA,Weinheim1414M.M.Shaw and B.M.Riederer Proteomics2003,3,1408–1417Preparative IEF devices include the“Rotofor”[48],pro-duced by Bio-Rad(Hercules,CA,USA),which is based on a system proposed by Bier et al.[49],the“Isoprime”from Amersham Biosciences(Uppsala,Sweden)based on a development by Righetti et al.[50]and the“Gradi-flow”from Gradipore,demonstrated by Corthals et al.[51].The Rotofor is a rotating chamber in which samples are fractionated in solution by IEF.However,this appara-tus has no separation barriers and fractions obtained are not well-resolved,with cross-contamination of proteins between fractionated pools.The Isoprime apparatus is a multicompartment electrolyser where each compartment is separated by a polyacrylamide gel membrane with a specific pH produced by Immobilines incorporated into the membranes.The Isoprime apparatus produces high quality fractions.However,it was developed primarily for large-scale separation of partially purified preparations, not for fractionation of crude extracts.Zou and Speicher [52]developed a solution IEF device based on a scaled-down volume version of the Isoprime apparatus for pre-fractionation of cell extracts,which also yielded well-resolved fractions.This has been further developed as the microscale solution or“nusol”IEF technique[53].A further off-gel IEF unit,consisting of a fluid-filled multiwell device placed on top of an IPG gel has been developed by Michel et al.[54].Proteins diffuse through the gel and into the solution of each well.This effectively increases the capacity of the IPG gel and provides protein at fractionated p I ranges in solution for further manipu-lation.The system allows resolution to as high as0.1pH unit.3.2Resolution of“elusive”proteinsThere exist several classes of proteins which have eluded resolution on2-D gel maps and thus represent some of the proteins which continue to remain beneath the tip of the“proteome iceberg”.Proteins fail to be resolved on2-D gels for a number of reasons.They either do not enter the first dimension gel,or they enter the gel but precipitate and do not migrate,forming streaks.Precipitation also makes transfer to the second dimension difficult.Other proteins may be present at such low levels that they escape detection or they may be highly unstable and degrade so rapidly that they can-not be detected,factors which may be counteracted by prefractionation or the use of protease inhibitors.Finally, some proteins may not be resolvable on2-D gels simply because their p I s fall outside the range of the first dimen-sion.The latter are exemplified by the very basic(p I.10) ribosomal proteins and histones.This problem was solved by developments which enabled extension of the p I extremes of IPG strips[55].The increased reverse electroendosmotic flow in these strips necessitated the inclusion of isopropanol and glycerol in the reswelling so-lution[4].The failure of proteins to enter IEF gels may be due to sev-eral factors.One is size.The spectrins represent a group of large(280kDa)filamentous proteins which,when using IPG strips in the first dimension,are lost from the2-D map unless active instead of passive sample hydration into the IPG strip is carried out[33].Another property,which affects the ability of proteins to enter IEF gels or remain in solution during focusing is solubility.Proteins which resist solubili-sation in specific sample buffers are invariably hydropho-bic membrane proteins,nuclear proteins and proteins highly prone to aggregation such as tubulin and keratins. Improved solubilisation of keratins can be achieved by ensuring(and maintaining)maximal reduction of disul-phide bonds.This may be achieved by alkylation immedi-ately after reduction,or with the highly potent,non-charged reducing agent,TBP.Further improvements have been made by the inclusion of thiourea;in the absence of this strong chaotrope,tubulin and fibronectin are barely visible on2-D gels.The addition of zwitterionic amphiphilic surfactants(e.g.CHAPS or SB3–10)has also improved protein solublisation.In a study comparing the solubilisa-tion properties of various sample buffers,Rabilloud et al.[6]found that CHAPS-containing solutions consisting of urea and thiourea were more effective at solubilising high M r integral membrane proteins than SB3–10-containing solutions.However,since SB3–10is not compatible with high levels of urea,the urea concentration in this case had to be limited to5M,whereas the CHAPS-containing solu-tion contained7M urea.Therefore,a direct comparison of the efficacy of the two surfactants was not made.A similar indirect comparison was carried out by Molloy et al.[56]who compared the solubilisation efficacies of a buffer containing2%CHAPS and2%SB3–10with a buffer containing1%ASB14.The remaining components of both buffers were essentially the same,except that the CHAPS/SB3–10buffer contained5M urea and2M thiou-rea,whereas the ASB14buffer contained7M urea and2M thiourea.They found that the ASB14-containing buffer produced larger spots on the2-D gels,indicative of more protein.However,this improved yield may also be a result of the increased urea concentration used in conjunction with this surfactant.The alkyl aminosulphobetaine,ASB14,used in conjunc-tion with urea and thiourea,resulted in the appearance of several integral membrane proteins not previously detected on2-D maps[57].Nouwens et al.[58]directly compared two sample buffers(both containing7M urea, 2M thiourea,0.5%carrier ampholytes and2m M TBP), one buffer additionally containing2%CHAPS and2%2003WILEY-VCH Verlag GmbH&Co.KGaA,Weinheim。