Solubilization of Proteins

download Solubilization of Proteins

of 17

Transcript of Solubilization of Proteins

  • 8/6/2019 Solubilization of Proteins

    1/17

    Elecrrophores is 1996, 17, 813-829 Solubilization of proteins 813

    ReviewThierry Rabilloud Solubilization of proteins for electrophoretic analysesD B M S , CEA, Grenoble, France

    Contents Another general problem is linked to the requirements122.12.22.2.12.2.22.2.32.2.42.2.52.32.433.13.1.13.1.23.23.2.13.2.245

    Introduction ...........................General principles ....................Rationale of solubilization . . . . . . . . . . .Removal of interfering substances . . .Salts ..................................Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Nucleic acids .........................Polysaccharides ........................Other compounds .....................The case of disulfide bonds . . . . . . . . .The problem of protease action . . . . .Specific solubilizations for electro-phoretic separations . . . . . . . . . . . . . . . . . .Zone electrophoresis . . . . . . . . . . . . . . . . . .Denaturing zone electrophoresis . . . . .Native zone electrophoresis . . . . . . . . . .Isoelectric focusing . . . . . . . . . . . . . . . . . . .Native isoelectric focusing . . . . . . . . . . . .Concluding remarks . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . .Denaturing isoelectric focusing . . . . . . .

    81381381 381581581581681781781781982 082182282382 482 482 6827828

    1 IntroductionElectrophoresis is now increasingly used to resolve hun-dreds of proteins for analysis by amino acid composition,microsequencing, and more recently mass determinationwith mass spectrometry, thanks to the scaling down ofthese techniques. Most often, the high-resolution separa-tion needed can only be performed on polyacrylamidegels, and sometimes by two-dimensional gel electro-phoresis only. As for almost all the analytical techniquesused in protein chemistry, the proteins to be analyzedmust be (i) extracted from the biological sample,(ii) freed from any substances which could interfere withthe analytical technique or the subsequent processes,and (iii) kept in solution during the whole separationprocess. These three steps represent the solubilizationprocess, which may vary greatly from sample to sampleor from one type of electrophoretic technique to another.Correspondence: Dr. Thierry Rabi lloud, DBMS , CEA-Gren oble , 17 ruedes martyrs, F-38054 Gr enob l e Cedex 9, France (Tel: +33-7688-3212;Fax: +33-7688-5155; E-mail: [email protected])

    Nonstandard abbreviations: C12E8: Dodecyl octa(ethy1ene glycol);3,4 DCI: 3,4 dichloroisocoumarin; E64: N-[N-(L-3-transcarboxyrane-2-carbony1)-L-Leucyll-agmatine; N D S B : non detergent sul fobeta ines ;NDSB2Ol: pyridinio propane sulfonate; S B 3-14: tetradecyl dimethyl-ammonio propane sul fonateKeywords: Solubilization I Two-dimensional polyacrylamide gel elec-trophoresis / Interfering substances

    of the structural analyses methods. Although peptidemass fingerprinting and amino acid composition can beperformed at a scale that is close to the one of purelyanalytical separations, other techniques such as microse-quencing of mass analysis by electrospray will stillrequire microgram amounts of the protein of interest.This means that even the final electrophoretic separationmust be carried out on a larger scale than a purely ana-lytical one. This in turn gives rise to new problems, suchas the elimination of interfering substances, which canoften be neglected in analytical separations. A section ofthis review will therefore be dedicated to the problem ofremoval of interfering substances.The aim of this review is, however, not to list the incred-ibly high number of solubilization processes that havebeen described in the literature. Such a task would be oflimited help to the reader and almost impossible to carryout properly for the author. Instead I would like to con-centrate on the rationale of solubilization, and then todescribe the main solubilization protocols correspondingto the main electrophoretic techniques and their prin-cipal variants, with selected references to give examplesof the most interesting variants in my opinion. In mostcases structural analyses (e.g. microsequencing) will beperformed o n polypeptides, which means that all thebonds linking the polypeptide chains of the proteinsmust be broken prior to the analysis. This allows the useof denaturing solubilization in most cases, so that thefractionation prior to primary structural analyses willonly use parameters that do not require the native three-dimensional conformation, such as the isoelectric pointor the polypeptide chain molecular mass. However, non-denaturing solubilizations can be required; for example,if the activity andlor conformation is needed for purifica-tion prior to the final separation steps; or if the struc-tural analysis needs to be performed on a complete pro-tein (e.g. mass spectrometry on a total protein). Someexamples include the immunopurification with an anti-body directed against a conformational epitope, or thesolubilization of a multimolecular complex as a whole totake advantage of its huge but precise size to separate itfrom the bulk of smaller proteins [l].This explains whythis review will try to cover both denaturing and nonde-naturing solubilization methods.2 General principles2.1 Rationale of solubilizationSolubilization can be defined as a process breaking theinteractions between the substances to be analyzed andinterfering substances, eliminating the interfering sub-

    0 CH Verlagsgesellschaft mbH, 69451 Weinheim, 1 9 9 6 0173-0835/96/0505-0813 $10.00+.25/0

  • 8/6/2019 Solubilization of Proteins

    2/17

  • 8/6/2019 Solubilization of Proteins

    3/17

    Electrophoresis 1996, 17 , 813-829 Solubilization o f pro te ins 815from the other molecules with which they are able tointeract, but can also lead to denaturation, i . e . disruptionof the three-dimensional structure of the proteins. Theratio between both events will depend on the relativestrength of the intermolecular and intramolecular inter-actions and of the disruptive capacity of the solubiliza-tion medium. Second, it must be kept in mind that allthe noncovalent forces keeping the molecules togethermust be taken into account with a comparative look atthe solvent. This means that the final energy of interac-tion depends of the interaction p e r s e and of its effectson the solvent. If the solvent parameters are changed(dielectric constant, hydrogen bond formation, polariza-bility, e tc . ) , all the resulting energies of interactions willchange. This explains why chaotropes, which alter all thesolvent parameters, exert such profound effects on alltypes of interactions. For example, by changing thehydrogen bond structure of the solvent, chaotropesdecrease the energetical penalty for exposure of apolargroups and therefore favor the dispersion of hydro-phobic molecules and the unfolding of the hydrophobiccores of the proteins [6].As a conclusion, apart from the disruption of disulfidebonds, which will be described later, solubilization canbe described as the way to disrupt the noncovalentforces ensuring the cohesion of supramolecular com-plexes, for example by the means described in Table 1,so that these complexes will be broken to the desiredextent. If the three-dimensional conformation of the pro-tein is to be kept native, there will be a difficult balanceto achieve between a sufficient breaking of the unde-sired interactions and keeping the cohesive interactionsin terms of protein three-dimensional structure. Owingto the key role of hydrophobic interactions in the three-dimensional protein structure [4], this explains why thecases in which these interactions are to be broken arethe most difficult, and, for example, why nondenaturingsolubilization of membrane proteins is so difficult. Theprocess will, of course, be even more difficult if multi-molecular complexes are to be kept together during thesolubilization and the separation process. The solubiliza-tion protocol will therefore depend on the requirementsof the purification method (in our case, electrophoreticmethods), on the choice of conditions (native or dena-turing), and on the problems brought by the interferingnon-proteinaceous substances present in the biologicalsource of macromolecules. These interfering substancesmust generally be removed prior to separation of theproteins by electrophoresis, as they often interfere withthe electrophoretic processes. This selective removal is adifficult part of the solubilization process; the followingsection of this review will therefore be devoted to thisproblem.

    2.2 Removal of interfering substancesInterfering substances are the compounds that interferewith the solubilization and/or the electrophoreticprocess, either by binding to the proteins to be analyzed,which often prevents a proper separation, or by pre-venting the electrophoretic process p e r s e . If proteins areconsidered the valuable analytes of the sample (which isthe case within the scope of this issue) everything in the

    cell but proteins can be considered as an interfering sub-stance. Some of them (e .g . , coenzymes, hormones,simple sugars, nucleotides) are either so diluted thatthey do not give any problem or neither interact with theproteins nor with the electrophoresis (e .g . nonreducingsimple sugars). Unfortunately, many other classes ofcompounds give rise to an interference, and themethods to eliminate them will be examined below.2.2.1 SaltsIn most cases, salts do not interfere by a strong bindingto the protein, but rather by disturbing the electro-phoresis process. In the cases where some proteins havea high affinity for an ion (e .g . calcium-binding proteins),the use of ion chelators or denaturation often relievesthe problems. In the extreme cases where the ion isstrongly coordinated (e .g . iron in the covalent heme ofsome cytochromes, zinc in the zinc finger structures) thestrength of the coordination is so high that the molec-ular species which is analyzed is an ion-protein complexof defined stoichiometry, which is generally trouble-free.In general, high amounts of salts are present in halo-philic organisms or in some biological fluids ( e . g .urine,sweat, and, to a lesser extent, plasma and spinal fluid).The general method used to remove salts is based ondialysis, since salts have much lower molecular massesthan protein. Devices which both concentrate the pro-tein and dialyze it (vacuum dialysers, centrifugeabledevices, etc.) are often used (e .g . in [7]). The major prob-lem which may arise is loss of protein by adsorptiononto the dialysis membrane, or loss by diffusion throughthe membrane for low molecular weight proteins. Theformer problem can be solved in some cases by dena-turing the proteins in the presence of detergents, whichwill greatly reduce the adsorption to the membrane.However, it must be kept in mind that micelles aregenerally large enough to be retained by the membrane,so that any concentration of the sample will lead to aconcomitant increase in the concentration of the deter-gent. Alternatively, if denaturation is tolerated, bothproblems can be solved by precipitation with TCA inwater and resolubilization in a medium convenient forelectrophoresis (e .g . in [8]). Other promising methods arebased on the precipitation of proteins with dyes [9, 101.These methods are convenient substitutes of TCA preci-pitation for the elimination of salt. Owing to the selec-tivity of the protein-dye interaction, they should allowselective precipitation of proteins, and therefore an easyremoval of a wide range of interfering compounds. Therecent introduction of these methods has not yet per-mitted full evaluation of their potential.

    2.2.2 LipidsLipids give two kinds of problems depending of theirsupramolecular structure, i . e . as monomers or as assem-blies ( e . g . membranes and vesicles). As monomers, theycan bind to some proteins, usually lipid carriers. Such abinding can alter the characteristics used for the electro-phoretic separation (pl, molecular mass) and give rise toartifactual heterogeneity. This problem is generally a

  • 8/6/2019 Solubilization of Proteins

    4/17

    816 T. R a b i l l u u d Elerrrophores is 1996, 17 , 813-829minor one, which is easily solved by the use of deter-gents in a denatur ing medium for the most tenaciouscomplexes . The problem becom es much more ser iouswhen supramolecular assemblies of l ipids (mem braneand derivatives thereof) a re present. As a basic rule, thepresence of detergents is the solution of choice to dis-rupt th e m em brane s, solubilize th e lipids, delipidate andsolubi l ize the proteins bound to those membranes orvesicles. Conseq uently, many reviews have been writtenon the properties and uses of detergents and the readeris referred to some of them [ll-141, as well as to somecomparative work on the efficiency of various detergentson membranes [15]. However, as stated earlier in thisreview, detergent acts by diluting the lipids into themicelles. A problem will therefore arise for samples withhigh lipid levels. In this case, there will soon be an ina-dequacy between the amount of lipids present in theamount of sample required to prepare the protein forthe structural analyses and the amount of detergent thatcan be used in the desired sample volume (insufficientdetergency). Two solution s may be envisioned. The firstis to scale u p the separation, and therefore dilute thesample, so that a correct detergency can be achieved.This is l imited by the volumes which can be loaded onthe electrophoretic gels . The second solution is to carryout a chemical delipidation on the sample prior to reso-lubilization of the proteins in the presence of deterg ents.Delipidation is achieved by extraction of the biologicalmaterial with organic solvents [16], generally a mixture[17], and often containing chlorinated solvents [18]. Suchmedia are often strongly denaturing, so that the subse-quent resolubilization of the proteins must be made indenaturing conditions. However, more conventional andsometimes nondenaturing protein precipitation proto-cols, for examp le with e thano l or ace tone, often providea partial but useful delipidation [19, 201.Generally speaking, these media based on organic sol-vents remove excess lipids efficiently. However, a severeloss of proteins may be experienced , either becausesom e proteins are soluble in organic solvents [17], or per-haps because the precipitated proteins do not resolubi-lize. Special attention m ust be paid to th e final removalof the organic solvents prior to resolubilization. If thesolvent is not efficiently rem oved, em ulsion p roblems orprecipitation by the rem ainin g solvent may arise. If theprecipitated protein pellet is dried too extensively inorder to remove the solvent completely, a t ight and drypellet - mpossible to resolubilize even in media of highdenaturing and solubilizing power - appears, with ex-tremely severe losses. This leads to th e picture t hatachievement of a proper and reproducible delipidation-solubilization cycle is difficult as soon as delipidation byorganic solvents is required. As a practical rule, theprocess becomes more and more difficult as the solventused becomes less and less miscible with water. Conse-que ntly, partial delipid ation with alcohols is often easie rthan with ac etone , which is itself much easier than withchlorinated- or ether-based solvents.2.2.3 Nucleic acidsSeveral types of problems are encountered because ofnucleic acids. First, nucleic acids behave as polyanions

    and are therefore able to bind many proteins throughelectrostatic interactions. Second, this problem is evenmo re sev ere when separation with isoelectric focusing isto be performed, as nucleic acids also bind carrierampholytes to give complexes [21], that also bind pro-teins and focus to give completely artifactual results witha high amount of streaking [22]. Third, nucleic acids(especially DNA) are very long molecules that are ableto increase the viscosity of the solutions considerablyand also to clog the small pores of the polyacrylamidegels used to separate the proteins. Wh en n ative, low-ionic-strength extractions are performed, the nucleicacids stay compacted as protein-containing complexeswhich can easily be removed by low to medium speedcentrifugation (at most 10 00 0 g ) . When high salt ordenaturing extractions are performed, these complexesare dissociated and the nucleic acids swell in the solu-tion, causing the problems described earlier .For these reasons, removal of nucleic acids is required,unless they are present at very low con centrations [23]. Itcan be effected by several m ethod s. The first m ethod isdigestion by nucleases, initially by a mixture of RNAsesand DNAses [24], followed in some occasions byremoval of th e digestion products (oligonu cleotides) byTCA precipitation [25]. As with most of the enzyme-based removal methods, the main drawbacks are l inkedto the parallel action of the proteases contained in thesample, thereby degrading the proteins , and the addi t ionof ex traneo us proteins (th e nu cleases). The first draw-back can be partially alleviated by digestion with S l nuc-lease in a urea-containing medium [26], although it hasbeen shown that some proteases are even sti l l active inthis medium [27]. In fact, the mo st efficient method s usecentrifugation to get rid of the excess nucleic acids.When SDS-electrophoresis is planned, DNA and largeRNA s are only a problem b ecause of their large size andviscosity, which give rise to gel clogging [28]. Ultracentri-fugation has been used for years and has shown to beefficient in eliminating these large nucleic acids [29, 301.When isoelectric focusing is planned, advantage can betaken of the form ation of nucleic acid-carrier ampholytecomplexes to eliminate them by ultracentrifugation [25].A last problem remains, however: some proteins sti l ls tick to nucleic acids even in the presence of high con-centrations of urea [31]. These proteins can be solubi-lized either with competing cations such as protamine[31] or lecithins at acid pH [32], or the extraction pH canbe increased so that all the proteins will behave asanions and will be repelled from the anionic nucleicacids. To avoid overswelling of the nucleic acids, whichdecreases the efficiency of the subsequent removal byultracentrifugation, this increase of pH can b e m ediatedby the addition of a basic polyamine (e.g. spermine [33]),which will precipitate the nucleic acids. Ultracentrifuga-t ion-based methods could be seen as r isky methods ,since large proteins could also sediment during theremoval of nucleic acids, especially if this removal has tobe complete and include small nucleic acids. I t must,however, be kept in mind that extraction is carried outeither with reage nts that de crease the buoy ant density ofproteins ( e . g . SDS) , or with reagents that increase thedensity of the solvent (salt, urea). This will lead todecreased sedimentat ion of the proteins compared to

  • 8/6/2019 Solubilization of Proteins

    5/17

  • 8/6/2019 Solubilization of Proteins

    6/17

    818 T. Rabi l loud Electrophoresis 1996, 17 , 813-829Table 2. Removal of interfering compoundsMethods Salts Lipids Nucleic Polysaccharides Pigmentsa Proteinacids recovery

    SSV

    Detergent - S/Lc Sb) V b ) --+L S VLltracentrifugation -Precipitation with-S Vomplex ions - -

    TCA S L - V V STCA / solvent wash S S - V S VAmmonium sulfate +d l Se SO VS VP) SSolventh - - S VS

    Abbreviations: S, satisfactory; V, variable; V,, variable with the size of the compound; L, limited;Lc, limited, depending on the concentration of the compound; - inefticienta) Pigments, terpenes, polypbenol and other related compoundsb) Efficient on ly with cationic detergents (inducing in fact a precipitation), with centrifugation toc) Some lipids may form an upper layer upon centrifugation; in favorable cases, this layer can bed ) Salts are removed, but replaced with residual ammonium sulfate, which can be removed with 70%e) Flotation of lipids is often induced.f) Efficient with a two-step procedure: dissociation of the proteins from nucleic acids in 0.6-0.8 Mammonium sulfate, ultracentrifugation to remove them, then quasi-saturation in ammonium sulfate

    to precipitate proteins.

    remove the precipitateremoved.ethanol.

    g) Variable, the solubility in concentrated ammonium sulfate not being predictable.h) Including phenol/ammonium acetate precipitation [38].

    S-

    +R- SH

    I

    H SS-S-R

    +R - S H

    T

    !--/OHR-S-S-R A O H

    Figure 2. Mechanism of disulfiede (reduction) with free thiols. Leficolumn: reduction with free monothiols ( e . g . mercaptoethanol). Rightcolumn: reduction with cyclizable dithiols (e.g. DTT).because of the residual persulfate, or reaction productsthereof [44, 451. This process can be blocked either byusing washed gels in which the spurious, oxidizing chem-icals have been washed away, or, in zone electrophoresis,

    by a continuous influx of thiols from the electrodebuffer [46]. Indeed, thiols are very weaks acids (pK, of ca .9), so that a fraction of the molecules bear a negativecharge (in the case of mercapthoethanol or DTT) andwill migrate into the gel and reduce the oxidizing com-pounds contained in the gel, thus maintaining areducing environment in the gel. Variants of this processuse sulfonylated thiols such as mercapthoethane sulfonicacid [47], or cysteamine (a positively charged thiol) foracidic gel systems [48].Another, more drastic, way to prevent reoxidation is toalkylate the reactive free thiols generated by proteinreduction into nonreactive thioethers. Such a reaction isgenerally achieved by nucleophilic substitution on haloa-cetyl derivatives, generally iodoacetic acid or iodoaceta-mide [49], or by nucleophilic addition onto an activateddouble bond, as for substituted maleimides [50] , inylpy-ridine [51] or even acrylamide [52]. This alkylationprocess is generally compatible with post-electrophoreticprimary structure analyses, such as amino acid analysis,microsequencing or mass spectrometry. In the latter case,however, care must be taken with the mass incrementsbrought by the alkylating reagents. Moreover, the reac-tion specificity for thiols is often not absolute, and otherprotein nucleophilic groups can also react, as the&-aminogroups of lysine, with the most reactive agents,such as iodoacetic acid. This can, however, be controlledin most cases by the careful choice of the pH of the reac-tion [49]. When such an alkylation reaction is performed,another drawback of thiols as reducing agents is broughtto light. Indeed, the free thiols that are needed to drivethe thiol-disulfide equilibrium will also react with thealkylating reagent, which must therefore be present inexcess with respect to the total thiols (protein plusreducing agent). This precludes the use of mercapto-ethanol, and even with DTT one comes to a point wherethe alkylating reagent is used close to its solubility limitand where a considerable amount of byproducts (HI in

  • 8/6/2019 Solubilization of Proteins

    7/17

    Electrophoresis 1996, 17 , 813-829 Soluhi l iza t ion of pro te ins 819

    the case of iodoacetyl derivatives) are generated. Thelatter drawback is, however, not observed for additionalreagents (NEM, vinylpyridine, acrylamide). Anotheradditional drawback of thiols as reducing agents lies inthe fact that some proteins seem resistant to reductionby mercaptoethanol or DTT. One example is the calci-um-binding protein SlOOa (J. Baudier, personal commu-nication). This is due to the fact that the thiol group ofthe protein is so reactive that it will either react with thefirst thiol reducing equivalent (see Fig. 2) but not withthe second one, leading to an adduct, or even not reactwith the thiol reagent.Owing to these drawbacks, additional reducing reagentshave been investigated, which would lead to reductionby a stoichiometric process and not by an equilibriumdisplacement process. Many of the agents used inorganic chemistry to achieve disulfide reduction are notof practical use in biochemistry (borohydrides, sodiumin acidic medium, etc.). However, trivalent phosphorusderivatives have proved to be very promising for reduc-tion of disulfides under biochemical conditions. Of these,phosphines have been shown to be efficient, while phos-

    phite esters are of marginal interest. Phosphines arecompounds in which the phosphorus atom is linked tothree simple hydrocarbon groups. The lowest membersof the class (e.g. trimethylphosphine) are highly reactiveand pyrophoric, while higher members (e.g. triphenyl-phospine) are safer to use but of very limited solubilityin water. A good compromise is tributylphosphine,which was the first phosphine used for disulfide reduc-tion in biochemistry [53]. These compounds, which reactwith disulfides as shown in Fig. 3, show many advan-tages. First, the reaction is stoichiometric, which, in turn,allows the use of a very low concentration of thereducing agent (a few mM). Second, these reagents arenot as sensitive as thiols to dissolved oxygen. Third,because of the limited concentration of the agent, alkyla-tion is much easier to perform. Phosphines interferewith alkylation with iodoacetyl derivatives. However, atypical reduction-alkylation will involve 2 mM tributyl-phosphine and 4 to 20 mM alkylating agent [54], insteadof 50 mM DTT and 150 mM alkylating agent. Moreover,phosphines do not interfere with compounds containingdouble bonds, so that simple and convenient one-stepprotocols contacting the protein with 2 mM phosphineand 10 mM alkylating agent (final concentrations) for30 min at room temperature and pH close to neutralitygive total reactions.Some drawbacks can be found for the use of tributyl-phosphine. The reagent is volatile, toxic, has a ratherunpleasant odor, and must be brought into water with

    Figure 3 . Mechanism of disulfide reduc-tion with phosphines (e.g. tributylphos-phine). Although the overall mechanismis known, the degree of concertation inthe electron transfer process betweenwater, the disufide and the phosphineis speculative (intermediate betweenB u ~ P = O brackets).

    the help of an organic solvent. In the first uses of thereagent, propanol was used as a carrier solvent at ratherhigh concentrations ( 50%) [53]. However, it was foundthat DMSO or DMF are suitable carrier solvents, whicheasily allow reducing the protein with 2 mM tributylphos-phine [54]. All these drawbacks have disappeared withthe introduction of a water-soluble phospine, tris(carbo-xyethy1)phosphine (available from Pierce), for which 1 Maqueous stock solutions can be easily prepared andstored frozen in aliquots. In addition to their superiorityon thiol reagents, as soon as subsequent alkylation isperformed, phosphines have the additional advantagethat they reduce all protein disulfide bonds, even thoseresistant to DTT (as the SlOOa protein; Jacques Baudier,personal communication), and that they do not yield anyadduct formation, as has been described for mercaptoe-thanol [45]. The use of phosphines as reducing agentsshould therefore be systematically envisioned as long astotal reduction of all the accessible disulfides is planned,which is almost always the case when subsequent pri-mary structure analyses are planned.

    2.4 The problem of protease actionProteases present in the sample are everything butfriends for the protein biochemist, especially when struc-tural analyses are planned. Although proteolysis is animportant mechanism to mature and recycle proteins invivo, a great amount of artifactual proteolysis can occurwhen the tissue or cell is broken for solubilization,owing to the breakage of the compartmentalizationexisting in vivo. This proteolysis should be avoided by allmeans, since it can lead to severe artifacts and misfacts,for example if N-terminal sequences or mass determina-tions are looked for, or generate artifactual polypeptides,or decrease the yield for the desired protein. Proteasesmay be classified according to the amino acid whicheffect the catalytic attack of the peptide bond. This leadsto the distinction between serine, cysteine, and aspartylproteases. Other proteases (metalloproteases) need ametal ion to effect proteolysis. Destruction of this aminoacid or active site will therefore produce an efficient andirreversible means of avoiding proteolysis.In another approach to avoid proteolysis, advantage istaken of the specificities of action of proteases (e.g. pH).For example, lysosomal proteases are active at low pHand inhibited at high pH, which can be used in the solu-bilization process. Advantage can also be taken of theoccurrence of natural or synthetic peptidic proteaseinhibitors, which bind to and block the active site oftheir substate protease(s) (e.g. trypsin inhibitors such asa1 antitrypsin, Kazal- or Kunitz-type inhibitors, etc.).

  • 8/6/2019 Solubilization of Proteins

    8/17

    820 T Rabi l loud ElectrophuresiA 1996, 17 , 813-829The problem of protease action is of course most prom-inent when native solubilization is performed, as all theproteins, including proteases, are native and have theirmaximal activity. In most animal cells and tissues, themain problem arises from lysosomal proteases. Sincethese proteases are most active at low to medium pH,solubilization at a pH as high as possible will greatlyhelp in preventing proteolysis. However, this is usuallynot sufficient, and cocktails of inhibitors are almostalways added to the solubilization medium. In general,these cocktails contain both peptidic and irreversibleinhibitors [55]. The most popular irreversible inhibitor isphenylsulfonyl fluoride (PMSF). This chemical reactswith the activated serine of the catalytic center of serineproteases and prevents it from playing its catalytic role,so that irreversible inhibition is obtained. This chemicalhas been used by a host of workers, with variablesuccess (e.g. [56, 571). Indeed, the use of PMSF is notwithout drawbacks. (i) Its solubility in water is ratherlow, so that it must be introduced with the help of acosolvent (generally ethanol or propanol). In spite ofthis, precipitation often occurs when PMSF is added tothe extraction solution, which decreases the active con-centration. This in turn leads to a severe decrease in pro-tease inhibition [58]. (ii) PMSF is unstable and degradesby spontaneous hydrolysis (half-life at room temperatureand pH 8 = 30 min). (iii) Some serine proteases seem tobe quite resistant to PMSF, as human trypsin 1 [59].Most of the drawbacks of PMSF can be alleviated byusing other irreversible inhibitors. DFP (diisopropyl fluo-rophosphate) is much more potent and water soluble,but is very toxic. The best compromise seems to be ami-noethylbenzylsulfonyl fluoride (AEBSF), a more stableand water-soluble analog of PMSF. This compound, asDFP, has the drawback of introducing an electric chargeto the molecule with which it reacts. This is not a pro-blem as far as protease inhibition is concerned. However,side reactions of these inhibitors on other nucleophilicgroups of the proteins (other serines, tyrosines orlysines) could introduce charge artifacts that are notcompatible with methods such as isoelectric focusing.Owing to its somewhat recent introduction, artifactsresulting from the use of AEBSF have not been de-scribed yet. Other irreversible inhibitors for serine pro-teases are those based on a reactive analog of the aminoacid recognized by the protease. Example of this classare tosyl lysyl chloromethyl ketone for the inhibition oftrypsin and tosyl phenylalanyl chloromethyl ketone forthe inhibition of chymotrypsin. These inhibitors are veypotent but also very specific, so that they do not inhibitefficiently the wide range of serine proteases generallypresent in a cell or a tissue.Irreversible inhibitors for cysteine proteinases are gener-ally thiol-alkylating agents, such as iodoacetyl derivativesor N-ethylmaleimide or mercury compounds. Aspartylproteases can also be irreversibly inhibited [39], but inhi-bition is effective only in the presence of copper ion,which precludes the use of metal chelators, which aregenerally added to inhibit the metalloproteases. To com-plement the action of irreversible inhibitors, whichcannot be used under all circumstances ( e . g . thiol-alky-lating reagents if native solubilization of a protein re-

    quires its free thiol groups), competitive, reversible, inhi-bitors are often added. Many of these inhibitors areshort peptides (e.g. pepstatin, antipain) or small mole-cules (e.g. benzamidine), which will not interfere withthe subsequent electrophoretic analysis. However, theuse of large molecules ( e .g . a2 macroglobulin) has alsobeen proposed [60], with the risk that this use will resultin artifactual polypeptides arising from the addition ofthe protein or of its partial degradation. Commonly usedinhibitors, which have been used either alone [56], or,more frequently, as various cocktails [39, 551 are listed inTable 3. Comprehensive reviews of protease inhibitionhave already been published [55, 611.Although dramatically decreased, the problem of pro-tease action is not abolished when denaturing solubiliza-tions are performed. Evidence of proteolysis after solubi-lization in 9 M urea [57] or SDS [62] has already been de-scribed. This is probably due to the fact that many pro-teases are resistant proteins, as shown by their ability towork in dilute SDS, used for example in peptide map-ping [63]. This means that their kinetics of denaturationin urea- or SDS-based solutions can be slow enough toallow them to work for a nonnegligible time, while mostof the other cellular proteins are already denatured andtherefore expose a maximal number of proteolytic sites.This problem of proteolysis in denaturing solutions willof course strongly depend on the concentration of pro-teases in the sample, and seems to be more important inplant tissues [57]. This work also demonstrated that addi-tion of protease inhibitors was of weak, if any, efficiencyto solve this problem [57]. In such difficult cases, theonly solution is to increase the denaturing power of thesolubilization process as much as possible. For musclesamples, where the degradation of the giant proteinsnebulin and titin is a problem [62], the use of a urea-thiourea denaturing solutions has been proposed [46,62].This solution was thought to penetrate faster into themuscle, therefore blocking the action of endogenous pro-teases faster. For plant samples, which are very rich inproteases, solubilization in boiling SDS has been pro-posed [64]. In this case, the thermal denaturation syner-gizes the SDS denaturation and affords a faster inactiva-tion of proteases. Another solution is to homogenize thesample in dilute TCA [65] or in TCA in acetone [42],which inactivates and precipitates all proteins, includingproteases almost instantaneously. Subsequent resolubili-zation of the protein precipitate in an SDS buffer [65] ora urea-containing buffer [42], does not seem to yield anyreactivation of the proteases. Such procedures are prob-ably the only efficient ones for tissues very rich in pro-teolytic activities.

    3 Specific solubilization for electrophoreticseparationsKeeping in mind the general principles and constraintsexposed above, the solubilization procedures used priorto electrophoretic separations will also depend on theconstraints introduced by the separation method itself. Iwould therefore like to discuss the solubilization proce-dures which can be used for the main types of electro-phoretic separations, i.e. zone electrophoresis on the one

  • 8/6/2019 Solubilization of Proteins

    9/17

    Electrophoresis 1996, 17 , 813-829 Solubil izat ion of proteins 821Table 3. Protease inhibitorsI r reversib le Inhibi tors Soect rum Useful concent ra t ionsAspartyl proteases Diazonorleucine methyl ester /Cu2 +Cysteine proteases N-ethylma leimideIodoacetate-iodoacetamidep-HydroxymercurybenzoateE64a)

    A E B S FT P C K b )

    Ser ine proteases PM SFT L C K ~ )3-4 DCF)

    Reversible inhibitorsAspartyl proteases PepstatinCysteine proteases AntipainSerine proteases AntipainLeupep t i nLeupeptinAprotinin

    Chymosta t inBenzamidineElastinalPhosphoramidonMetalloproteases EDTA

    Gene r a lGene r a lGene r a lGenera lGene r a lGenera lTrypsin-likeChymotrypsin-likeGeneral

    Pepsin, cathepsins,reninPapain-likeTrypsin-likeTrypsin-likeTrypsin-likeTrypsin,chymotrypsin,kallikreinsChymotrypsin-likeTrypsin-likeElaslase-likeGene r a lGene r a l

    2-10 mM1-10 m M1-10 mM0.5-10 vg/mL0.1-1 mM0.4-4 mM20-100 pg/mL70-100 pg / mL5-200 p~

    1-5 mM

    2-100 pg/mL2-20 pg/mL2-20 pg/mL2-20 pg/mL2-20 pg/mL1-10 pg/mL

    5-20 pg/mL5-50 pg/mL10-500 p~

    1-3 mM0.5-5 mM

    a) E64: N-[N-(~-3-transcarboxyrane-2-carbonyl)-~-leucyl]-agmatineb) To syl-~- lysin e hloromethyl k eton e; inhibits also cystein, proteases o f similar sub strate specificityc) 3,4 D C I : 3,4 dichloro isocoumarinhand and charge-based separations such as isoelectricfocusing on the other hand. As for solubilization requi-rements, two-dimensional electrophoresis will behaveas the first dimension, which is generally isoelectricfocusing. Concerning the solubilization methods, themain difference between what is allowed for zone elec-trophoresis and what is allowed for isoelectric focusingcomes from the fact that it will not be possible to useagents modifying the charge of the proteins in the lattercase. This gives rise to a serious solubilization problem,as electrostatic repulsion beween molecules cannot bereinforced by manipulation of the charge of the mole-cule. Oppositely, this manipulation is often desired inzone electrophoresis to decrease the problems comingfrom different charge densities of the molecules andfrom the pH dependency of the net charge of the pro-teins. In view of what has been stated in the introduc-tion, both native and denaturing solubilizations will bediscussed for each main method.3.1 Zone electrophoresisThe solubility problems encountered in zone electro-phoresis arise from the necessary use of discontinuouselectrophoresis to achieve correct resolution. This meansthat a stacking process of the proteins takes place, sothat the resulting concentration of the proteins withinthe stack is very high. Since the proteins which are separ-ated by zone electrophoresis migrate in the same direc-tion (the others being lost in buffers), they all have anelectric charge of the same sign; electrostatic repulsionthus takes place and decreases aggregation. Unfortu-nately, the charge density on most proteins is not highenough to give rise to a strong electrostatic repulsion;aggregation therefore occurs at high concentrations by

    the means of other binding forces (hydrogen bonds,hydrophobic interactions, etc.). This results in separa-tions that are very sensitive to loading; this is a severeproblem for structural analyses, which often require highloads of proteins. As an additional problem, proteinshave widely different pls [ 6 6 ] .This means that at a givenpH, there will be anionic and cationic proteins. This maylead in turn to electrostatic interactions and thereforeprecipitation within the sample. In addition, zone elec-trophoresis will separate proteins with a given type ofcharge (anionic or cationic), so that proteins will be lost,because electrophoresis cannot be carried out at pHextremes where all the proteins would have the sametype of charge.The best way to solve these problems is to use agentsthat will bind to proteins and give them an additionalcharge. This means in turn that separation on the basisof charge will be lost, and that separation will mainlyoccur according to the molecular mass. This is often nota problem, since separation on the basis of charge ismuch more efficiently carried out by specialized tech-niques such as isoelectric focusing. The use of chargemodifiers, by conferring to the proteins an additionalcharge (generally a negative one) has several advantagesfor solubilization. First, it allows converting most, if notall, proteins to species of the same type of charge. Byreinforcing the electrostatic repulsion, this dramaticallyincreases the solubility of the proteins before and duringthe electrophoresis process. This also allows the separa-tion of most proteins with a single type of electro-phoresis (mainly by considering proteins as anions). Lastbut not least, charge modifiers can also dramaticallyincrease the repulsion between proteins and interferingcompounds (e .g . nucleic acids), which may considerably

  • 8/6/2019 Solubilization of Proteins

    10/17

    822 T. Rabilloud Electrophoresis 1996, 17 , 813-829facilitate the solubilization process. In fact, these highlydesirable properties have been used in most zone elec-trophoretic protocols, denaturing or not.3.1.1 Denaturing zone electrophoresisIn a first approximation, the world of denaturing zoneelectrophoresis is very simple and is contained in threeletters: SDS. The somehow magic properties of SDScome in part from its ability to bind proteins at a highmass ratio (ca. 1.4 g of SDS per g of protein) basicallyindependent of the amino acid composition andsequence of the protein [67, 681. This independence isthe key of the superiority of SDS over other detergentssuch as decane, dodecane or tetradecane sulfonate, ordecyl or tetradecyl sulfate. Indeed, the masdcharge ratioof the SDS-protein complexes will be independent ofthe nature of the protein, so that the resulting electro-phoretic separation will be based only on the size of theinitial molecule [69]. However, recent work has shownthat undecyl sulfate might well even be superior to SDS,while confirming the amazing superiority of S D S overthe other anionic detergents [70]. On second thought,the magic properties of SDS are those of many ionicdetergents. With its long, flexible alkyl tail, SDS is ableto contract hydrophobic interactions with all combina-tions of amino acids, which leads to massive unfolding,and thus denaturation, of proteins. However, not alldetergents with C12 linear alkyl tails are so effective,dodecyl maltoside, Tween 20 and C12E8 being very milddetergents. The action of SDS is in fact largely due to itsionic head, which can break ionic interactions betweenproteins and, last but not least, drive an important elec-trostatic repulsion between SDS-protein complexes. Thisprevents reassociation of SDS-protein complexes, andkeeps them soluble even at the very high concentrationsencountered in gel electrophoresis. Other desirable prop-erties of SDS come from the fact that its ionic head isderived from a strong ion, so that the presence of thecharge is guaranteed in the pH interval 2 to 12, wheremost of the biochemical separations take place.To ensure maximum binding of SDS and proper denatu-ration of proteins, S D S has to be present in slight excessover the calculated ratio (say at least 1.5 g of SDS per gof protein), and a synergistic denaturation process has tobe applied. Indeed, for some proteins for which the intra-molecular cohesion is very important (and many pro-teases belong to this class), SDS alone may not be ableto disrupt the structure of the protein, but will only coatthe outside of the protein. An important negative charge,but only partial, if any, denaturation will be obtained.Healing (in SDS), as commonly described [71], inducesthermal denaturation and total exposure of the proteinto the solvent, so that SDS can bind to all parts of theproteins. The charge and intermolecular electrostaticrepulsion induced by SDS binding generally preventsthe denatured proteins from reaggregating and precipi-tating. However, certain membrane proteins precipitateupon heating in SDS, while they remain soluble withoutheating (J . L. Popot, personal communication). In theseproteins, which are rich in hydrophobic residues, SDSbinding to the unfolded protein might not be sufticientto overcome the host of hydrophobic interactions

    between molecules, which eventually lead to reaggrega-tion. Heating in the presence of SDS can be replaced,with almost equal efficiency, by the use of SDS and ureaat room temperature [72], which avoids the proteincleavage sometimes observed by heating in SDS [72].The efficiency and simplicity of solubilization by SD Sfollowed by zone electrophoresis is shown by the enor-mous popularity of the method, as shown by the ca.9000 citations of Laemmlis paper [71] per year. However,there are some cases for which SD S is not optimal, andfor which alternative procedures have been searched.The most difficult case for SDS is represented by glyco-proteins. The hydrophilic glycan moiety reduces thehydrophobic interactions between the protein and SDS,while the negative charges of the glycan (induced forexample by sialic acid) induce a repulsive electrostaticinteraction between SDS and the protein, both phenom-ena preventing the correct binding of SDS. Other diffi-cult cases are represented by very acidic or very basicproteins ( e . g . histones [73], which also exhibit anabnormal binding of SDS. These cases do not generallyresult in a loss of resolution, but rather in misdetermina-tion of the molecular mass. This is usually not a problemin the planning of subsequent structural analyses, inwhich case electrophoresis is used as a separationmethod and not to determine molecular masses. How-ever, abnormal migration can result in smears (glycopro-teins) or decrease resolution (histones) or artifactualcomigration. The alternatives which have been describedtherefore deserve attention.The limitation of SD S arises from its negative chargeand maybe too short alkyl tail; the solution was to inves-tigate the use of cationic detergent with longer alkyl tails(C14 to C16). This led to the description of cationicdetergent solubilization and electrophoresis, several var-iants arising from the use of different detergents or gelsystems [74-761. Cationic detergents indeed offer manydesirable properties. They solubilize and denature pro-tein efficiently, as long as a synergistic denaturation byurea [76] or heating [75] is carried out, but they also pre-cipitate large polyanions, including nucleic acids andcharged polysaccharides, thereby facilitating the removalof these interfering substances and also the extraction oftheir bound proteins [32, 761.The main problem with cationic detergents arises fromthe subsequent electrophoresis. High resolution electro-phoresis implies the use of discontinuous electro-phoresis. Discontinuous electrophoresis of cations needsto find correct leading and trailing cations and correctseparation pHs, which are generally acidic [74-761. Theproblems thus arise from the fact that the conventionalacrylamide polymerization initiators, TEMED and per-sulfate, are poorly efficient at low pH [77], and that per-sulfate precipitates many cationic detergents [78]. Thisled the early workers to use other polymerization sys-tems, using either riboflavin and light [75] or a mixtureof ascorbic acid, hydrogen peroxide and ferrous ion(Fentons reagent) [76] or even light and uranium [79].The toxicity of the latter system, and the erratic polymer-ization allowed by the former ones, have prevented thespreading of cationic detergent electrophoresis. It must

  • 8/6/2019 Solubilization of Proteins

    11/17

    Elecrrophoresis 1996, 17 , 813-829 Solubilization of proteins 823be emphasized here that erratic and poor polymeriza-tions are a real problem for the subsequent analysis ofproteins separated on polyacrylamide gels. Indeed,unreacted acrylamide can react with proteins [52, 80-821,inducing thiol and amine alkylation, which in turn resultin N-terminus blocking, lysine modification and adductsinterfering with mass spectrometry.Recent work on cationic electrophoresis, however, maybring this technique back to the front of the stage, andprovide a useful alternative when SDS electrophoresisdoes not perform well. The first solution, which has beendescribed, is to carry out electrophoresis at neutral pH,where standard TEMED-persulfate polymerization cantake place. This is possible by the careful choice of thebuffering (Tricine), leading (sodium), and trailing (argi-nine) ions [83]. Upon testing this protocol, I found it togive a somewhat decreased resolution compared to olderones (e.g. [75]), maybe because of the interferencebetween persulfate and cationic detergents. .In addition,electrophoresis at neutral or slightly basic pH is not tobe favored, as the side reactions between proteins andfree acrylamide increase with pH [84]. In my opinion,cationic detergent electrophoresis should be reexaminedin the light of the new photopolymerization systemintroduced by Righettis group [85]. This system hasmany desirable properties (reviewed in [86]), including ahigh polymerization efficiency over a wide range of pHdown to pH 3 [77], and insensitivity to the presence oforganic solvents or detergents [87, 881. This system wastested for zone electrophoresis in the cationic mode andfound to give very good results [89]. Minor problems,however, before cationic detergent electrophoresisbecomes as user-friendly as SDS electrophoresis are theabsence of good tracking dyes or difficulties in thestaining protocols; however, the compatibility of cationicdetergent electrophoresis with structural analyses (e.g.microsequencing), together with desirable propertiessuch as a high resistance to high loading, have alreadybeen nicely demonstrated [90].3.1.2 Native zone electrophoresisWhen compared to denaturing gel electrophoresis, thespecific problems of native electrophoresis are mainlythe result of solubility problems as soon as the proteaseproblem is considered as solved (see introduction of Sec-tion 3.1). As an additional problem, native electro-phoresis must be carried out at a pH close to neutrality.This implies in turn that many proteins will not bear astrong net charge, so that their migration in the gel ispoor, or even that many proteins will bear a charge ofopposite sign and will be lost. As most of the proteinshave a p l below 7 [66], native electrophoresis generallyseparates anions (e.g. Fig. 4 in [91]). However, a systemfor separation of cationic proteins at neutral pH wasrecently described [92].The best way to alleviate the problems inherent tocharge electrophoresis is to use charge-shift electro-phoresis, or derivatives thereof. The rationale of thistechnique is to try to recover the beneficial effects ofSDS without the denaturing power by using chemicalsthat will bind to proteins and give them a strong neg-

    Figure 4 . Native gel electrophoresis. Proteins, solubilized in 0.2 M Bis-Tris 0.1 M HC1 were loaded on a 10%T polyacrylamide gel, cast in0.15 M Tris, 0.1 M HC1 with a 4%T stacking gel, cast in 0.2 M BisTris,0.1 M HC1. Electrode buffer: 0.1 M Tris, 0.1 M taurine (this buffersystem, previously described for 2-D electrophoresis [33], has an ope-rative pH of 8 .6 ) . Electrophoresis was carried out overnight at 90V.The buffer system used here is more alkaline than the one describedby Schagger and Von Jagow [95], but is cheaper to us e and affords abetter pH compromise for native electrophoresis without charge-shifting agents. From left to right: carbonic anhydrase, 20 0 ng; soy-bean trypsin inhibitor, 10 0 ng; Ovalbumin, 100 ng; bovine serumalbumin: 100 ng; conalbumin, 100 ng. The following lanes are acidicperoxydoxin, 5 0 ng, under various disulfide bond reduction and alkyla-tion conditions. This shows that this protein is present as a dimer heldby disulfide bonds [91], but also by other noncovalent bonds. Proteindetection by silver staining [33].ative charge without denaturing them. Because of theresulting strong charge density, the intermolecular elec-trostatic repulsion is greatly increased, which dramati-cally improves the solubility, and therefore increases theloading capacity. Moreover, the pH dependency of theseparation will be greatly decreased. Even at neutral pH,all the proteins binding the charge-shifting agent willshow a strong negative charge, even those with a high PI,so that anion electrophoresis will be of almost universaluse. Last but not least, differential binding of the chargeshifting agent can lead to separation of proteins of sim-ilar molecular mass, by modulating the net charge of theprotein complexes.The first charge shifting agents used for electrophoresiswere detergents based on bile salts [93, 941. Because oftheir rigid polycyclic hydrophobic part, these detergentscan only contract limited hydrophobic interactions,which are generally not powerful enough to lead to pro-tein unfolding. They are thus generally considered non-denaturing detergents, although they are known to beable to inactivate some membrane proteins, maybethrough subunit dissociation. However, the binding ofmany bile salts seems to take place on a limited numberof sites on many proteins, so that they are weakly effi-cient as general charge shifting agents. A happy excep-tion seems to appear in taurodeoxycholate, which seems

  • 8/6/2019 Solubilization of Proteins

    12/17

    82 4 T. Rabil loud Electrophoresis 1996, 17, 813-829to be a powerful charge shifting agent, although its useis not recommended for multiprotein complexes [95].Another attempt was made by using sulfated alkyl oli-goethylene glycol detergents [96], and these proveduseful for the separation of photosynthetic proteins [97].Here again, the versatility of these chemicals as chargeshifting agents has not been sufficiently investigated.The most versatile charge shifting agent seems to beCoomassie Blue G-250, introduced by Schagger andco-workers [95,98]. Coomassie Blue binds to a very widerange of proteins (although there are still some excep-tions [98]) and gives them a strong negative charge,although the binding seems to be mild enough to allowthe preservation of sensitive multiprotein complexes[l , 951. This results in a good separation with fairly highprotein loads, while protein recovery from the gel andsubsequent analysis still appear feasible [99].In my opinion, the main interest of this technique ofnative electrophoresis, as far as structural analyses areconcerned, is twofold. The first case is when previouschromatographic separation of a desired activity do notyield a pure enough protein, so that the SDS patterndoes not allow choosing protein bands to carry out fur-ther analyses ( e .g . microsequencing). In this case, separa-tion of the proteins under the native mode, followed byelution of the bands, detection of the activity, and reelec-trophoresis of the activity containing band(s) on a dena-turing gel offers a powerful separation means. Thesecond favorable case is the one of large multiproteincomplexes. Native electrophoresis offers the opportunityof a simple and powerful separation of large complexesfrom the bulk of low molecular weight proteins andfrom smaller polymeric complexes [99]. An elegant two-dimensional electrophoretic separation can then bedesigned, with the first, native electrophoresis to sep-arate the large complexes, and the second, denaturing,electrophoresis to separate the subunits of each complex,which can then be submitted to structural analyses [98].A peculiar case is presented by some extremely hydro-phobic proteins such as the seed storage proteins. Theseproteins are soluble in denaturing detergent-containingmedia (SDS) and in hydroorganic mixtures [loo-1021,but not in conventional aqueous buffers. Their nativesolubilization and analysis must therefore be performedin hydro-organic mixtures containing at least 50 O/oorganic solvent. As standard polyacrylamide gelation sys-tems are not efficient under these conditions, this hasled to the design of new acrylic monomers [103-1051,which are rather hydrophobic, but which must absolutelybe used with such mixed solvents, and are therefore oflimited use. Another solution to the problem of carryingout electrophoresis in the presence of high amounts oforganic solvents could be afforded by a new photopoly-merization system, allowing polymerization of polyacryl-amide gels in the presence of high proportions oforganic solvents [87].3.2 Isoelectric focusing and two-dimensionalA s far as solubilization is concerned, two-dimensionalelectrophoresis parallels isoelectric focusing, as most

    electrophoresis

    high-resolution two-dimensional electrophoresis meth-ods use isoelectric focusing (generally denaturing IEF)as the first step. Some protocols [106-1081 have usedSDS-PAGE as the first dimension, which considerablydecreases solubilization problems. However, difficultiesare encountered in running and handling the large isoe-lectric focusing gels required for the second dimension.In addition, interfacing IEF gels with structural analysesis a difficult task, partly because of the interferencebrought about by carrier ampholytes on many occasions,and partly because of other problems such as difficultiesin blotting [109-1121. These problems have preventedthese variant methods from gaining widespread use. Itshould be also emphasized that interfacing between arestrictive SDS gel and an IEF gel is not easy [106, 1071and cannot be guaranteed to be a trouble-free process.The solubilization problems arising for isoelectricfocusing come from the fact that the electrostatic repul-sion between proteins, which is widely used for zoneelectrophoresis, cannot be used with this technique,which requires keeping the native charge of the protein.This raises problems at three levels: (i) During the initialsolubilization of the sample, there can be importantinteractions between proteins of widely different pZ and/or between proteins and interfering compounds ( e .g .nucleic acids). This yields poor solubilization of somecomponents. (ii) During sample entry into the focusinggel, there is a stacking effect due to the transitionbetween a liquid phase and a gel phase of a higher fric-tion coefficient. This stacking increases the concentra-tion of proteins and may result in precipitation. (iii) At,or very close to, the isoelectric point, the solubility ofthe proteins comes to a minimum. This can be explainedby the fact that the net charge comes close to zero, witha concomitant reduction of electrostatic repulsion. Thiscan also result in precipitation of the proteins. It shouldbe kept in mind that, apart for point (i), the main forcesresponsible for protein precipitation are hydrophobicinteractions and hydrogen bonds. The problem of pro-tein solubilization for and during isoelectric focusingthus comes to a minimizing of these interactions3.2.1 Denaturing isoelectric focusingFor denaturing isoelectric focusing, the magic SDS com-ponent of zone electrophoresis is replaced by anothermagic component: urea. Among many effects, urea hasbeneficial ones for solubilization for isoelectric focusing.First, it decreases considerably the strength of hydrogenbonds. Second, as it increases the water solubility ofhydrophobic compounds [6], it considerably decreaseshydrophobic interactions (see Section 2.1). This consider-ably decreases precipitation at the solution-gel interfaceor close to the isoelectric point. However, the use ofurea also induces denaturation, and thus exposure of thetotality of the hydrophobic residues of the proteins tothe solvent. This in turn increases the potential forhydrophobic interactions, so that urea alone is often notsufficient to quench completely the hydrophobic interac-tions. This explains why detergents, which can be viewedas specialized agents for hydrophobic interactions, arealmost always included in the urea-based solubilizationmixtures for isoelectric focusing. Of course, these deter-gents should not bear any net electrical charge, and only

  • 8/6/2019 Solubilization of Proteins

    13/17

    Electrophoresis 1996, 17 , 813-829 Solubilization of proteins 825nonionic and zwitterionic detergents can be used. How-ever, ionic detergents such as S D S can be used for theinitial solubilization, prior to isoelectric focusing, inorder to increase the solubilization and facilitate theremoval of interfering compounds. Low amounts of SDScan be tolerated in the subsequent IE F [113], providedthat high concentrations of urea [114], and of nonionic[113] or zwitterionic detergents [115], are present toensure complete removal of the SDS from the proteinsduring IEF. Higher amounts of SDS must be removedprior to IEF, for example by precipitation [41]. It musttherefore be kept in mind that SDS will only be usefulfor solubilization and for sample entry, but will not cureisoelectric precipitation problems.The use of nonionic or zwitterionic detergents in thepresence of urea presents some problems due to thepresence of urea itself. In concentrated urea solutions,urea is not freely dispersed in water but can form orga-nized channels (see [116]). These channels can bindlinear alkyl chains, but not branched or cyclic molecules,to form complexes of undefined stoichiometry calledinclusion compounds. These complexes are much lesssoluble than the free solute, so that precipitation is ofteninduced upon formation of the inclusion compounds,precipitation being stronger with increasing alkyl chainlength and higher urea concentrations. Consequently,many nonionic or zwitterionic detergents with linearhydrophobic tails [117-1191, and some ionic ones [32],cannot be used in the presence of high concentrations ofurea. This limits the choice of detergents to those withnonlinear alkyl tails or with short alkyl tails, which areunfortunately less efficient for quenching hydrophobicinteractionsThree types of detergents are therefore used for dena-turing isoelectric focusing in the presence of urea, whichmainly covers the field of two-dimensional electro-phoresis. The first type contains a phenyl ring in thehydrophobic part, and an oligooxyethylene polar head.The main compounds for this class are Tritons (mainlyTriton X-100) and Nonidet P-40 [24]. The presence ofthese detergents in a gel prevents subsequent blotting ofthe separated proteins [109]. The second type contains alinear alkyl tail and a very hydrophilic sugar-based polarhead. Detergents of this type include octyl glucoside[120] and lauryl maltoside [121], and seem to be moreeffective solubilizers than the first type. Moreover, atleast for monodimensional isoelectric focusing, thesedetergents give much less interference for blotting theseparated proteins than Triton-type detergents [1101. Thethird type is based on sulfobetaines as polar heads.Standard sulfobetaine detergents with linear alkyl tailshave received limited applications because they requirelow concentrations of urea. However, good results havebeen obtained in certain cases for membrane proteins[122-1241, although this type of protocol seems ratherdelicate [119]. The compatibility of sulfobetaines withurea can be greatly increased by introducing an amidogroup in the detergent molecule [119]. This allows theuse of much higher urea concentrations (7-8 M ), whichhas led to marked improvement of the patterns in cer-tain applications with membrane proteins [119]. How-ever, the lack of commercial availability of these chemi-cals has precluded extensive testing and use.

    The last, and most used, type of sulfobetaine detergentused for isoelectric focusing is represented by CHAPS,which combines a bulky, polycyclic tail with the sulfobe-taine head. This detergent has been shown to be muchmore efficient than Triton-type detergents [120, 1251, andhas gained wide popularity in the field of two-dimen-sional electrophoresis. However, it seems that the 2%concentrations suggested in the early publications [1251may be insufficient in some cases, and that higher con-centrations (3-5%) may be required, as shown by worksusing two-dimensional electrophoresis [33, 1201, or one-dimensional denaturing IEF and multicompartment elec-trophoresis [126]. Additional advantages of CHAPSinclude diminution of detergent interference in two-dimensional electrophoresis, thereby resulting in adecreased need for equilibration between dimensions, aslong as conventional, carrier ampholyte-based IEF is car-ried out [127]. In one-dimensional IEF, CHAPS also faci-litates subsequent protein blotting (C. Cochet, personalcommunication).Apart from the problem of inclusion compounds, themost important problem linked to the use of urea is car-bamylation. Urea in water exists in equilibrium withammonium cyanate, the level of which increases withincreasing temperature and pH [128]. Cyanate can reactwith amines to yield substituted ureas. In the case ofproteins, this reaction takes place with the a-aminogroup of the N-terminus and the &-amino groups oflysines. This reaction leads to artifactual charge heteroge-neity, N-terminus blocking and adduct formation detect-able in mas spectrometry. Carbamylation should there-fore be completely avoided. This can be easily done withsome simple precautions. The use of pure urea (analy-tical grade) decreases the amount of cyanate present inthe starting material. Avoidance of high temperatures(never heat urea-containing solutions above 37C) consi-derably decreases cyanate formation. In the same trend,urea-containing solutions should be stored frozen(-20C) to limit cyanate accumulation. Last but notleast, a cyanate scavenger (primary amine) should beadded to urea-containing solutions. In the case of isoe-lectric focusing, carrier ampholytes are perfectly suitedfor this task. Although the use of Tris has been advo-cated for the same purpose [129], I think that its sterichindrance and relatively weak reactivity (e .g . in otheraddition reactions with double bonds [SO]) make it apoor cyanate scavenger, compared to ampholytes oramino acids. If these precautions are carried out cor-rectly proteins seem to withstand long exposures to ureawithout carbamylation [126, 1301.Because of these drawbacks of urea, substitutes havebeen actively searched for, with little success up to now.One direction which has been explored is that of substi-tuted ureas [131-1331. These chemicals are much lessprone to cyanate formation, and are more powerfuldenaturants than urea itself [6]. However, little successhas been encountered, either because these chemicalsinterfere with acrylamide polymerization, or becausethese compounds are not more efficient than urea. Theirincreased denaturing effect comes from their increasedability to break hydrophobic interactions [6]. However,the solubilizing power of urea also comes from its ability

  • 8/6/2019 Solubilization of Proteins

    14/17

    826 T. Rabilloud Electrophoresis 1996, 17 , 813-829to break hydrogen bonds, and subsituted ureas seem tobe less efficient than urea in this area. This explains whysubstituted ureas have not superseded urea as solubi-lizing agen ts for IEF. Am ides have also b een tried assolubilizing agents for IEF, since they are also well-known protein denaturants [134]. Here again, l i t t lesuccess has been encountered. Formamide, a well-known denaturant of nucleic acids [135], could be auseful substitute, but its susceptibility t? alkaline hydrol-ysis with the formation of charged compounds preventsits use for IE F [136]. Sustituted am ides have also b eentried [131], but the g eneral interference of th ese so lventswith acrylamide polymerization has precluded furthertesting. However, the use of substituted ureas andamides could now b e reinvestigated, as the use of a newphotopolymerization system [85] allows one to poly-merize acrylamide in the presence of high concentra-tions of solvents [87].Currently, the lypical solubilization mixture prior todenatur ing IEF contains urea (> 7 M ), a detergent ( inmost cases nonionic or zwitterionic, or SDS if an addi-tional initial solubilizing power is needed), and a buffer,usually carrier ampholytes, which avoids any interferencewith the subsequent IEF and protects f rom carbamyla-tion. This mixture may, in som e cases ( e . g . m e m b r a n e o rnuclear proteins), not be powerful enough. This ismainly due to th e fact that the p H ran ges of most carrierampholytes used are centered on a neutral or weaklyacidic pH. The resulting pH of the solution is thereforeonly weakly acidic, so that a non-negligible amount ofproteins will behave as cations. This increases protein-protein or protein-nucleic acid complexes by electro-static interactions, thereby decreasing the solubilizationyield. In addition, proteases are still transiently active atthese acidic or neutral pH ranges [27]. To alleviate th eseproblems, the best solution is to perform the solubiliza-tion at alkaline pH. Proteases are inhibited at high pH[27], and all proteins will behave as anions, therebyincreasing the electrostatic repulsion between proteinsand nucleic acids or other proteins. This results in dra-matically im prov ed solu bilizatio ns, with yields very closeto those obtained with S DS [137]. The alkaline pH canbe obtained either by addition of a few mM of potassiumcarbonate to the urea-detergent-ampholytes solut ion[137], or by the use of alkaline ampholytes [127], or bythe use of a sperm ine-D TT buffer, which allows betterextraction of nuclear proteins [33]. In the latter buffer,spermine is used as a cyanate scavenger, and carrierampholytes of the desired range are added after solubili-zation and prior to loading on t he IEF ge l .Practical solubilization protocols prior to d enatu ring IE Fmay be summerized as follows: ( i) If no problems areencountered, use a s tandard urea-ampholyte-detergentmixture. Changing the detergent or mixing detergentscan considerably increase the solubilization efficiency forthe protein(s) of interest [121, 1251. (ii) If solubilizationis still poorly efficient, use an alkaline solubilizationbuffer of any of the thre e types previously de scribe d. D onot forget that ca rbonate will ente r into the focusing gel.This will improve the initial penetration of the proteins,but high amounts of carbonate (as in micropreparat iveruns with large amounts of samples) will distort the pH

    gradient [138]. (iii) If problems still remain, use a urea-carrier-ampho lyte-SDS buffer. Be aw are that this buffercan perform nicely on analytical runs and give very poorresults at the micropreparative scale due to SDS over-load. Precipitation of proteins to rem ove the excess SD S[41, 1391 may be necessary, with th e risk of lo sses of pro-teins at this step.3.2.2 Native isoelectric focusingAll the solubilization problems described in Section 3.2apply of course even m ore severely to native I E F than todenatur ing IEF because many general means used indena tur ing IEF - such as strong solubilizers (urea),powerful detergents or pH ex t r emes - cannot be used.The solubilization process prior to native IEF is there-fore a tedious testing process where combinations ofmild detergen ts, weak coso lvents such as ethylene glycol[140], are tested at the analytical scale for the solubiliza-tion of the protein of interest. As a general rule, thedetergents used in denatur ing IE F are m ild enough tobe used in native IE F. The on ly exception are simplealkyl sulfobetaines, which denature some proteins [15]but are efficient solubilizers, so that they deserve a tr ial .Within this family, I would recommend resting sulfobe-taines with C10 to C14 tails , either alone or in c ombina -tion with nonionic detergents [141].As nondetergent additives, polyols such as ethyleneglycol, glycerol, sorbitol or nonreducing sugars areuseful in some cases ( e . g . [140]). The use of reducingsugars should be avoided because of the possible Ama-dori reaction with the amino groups of the proteins,which could induce charge heterogeneity [1421. Othernondetergent additives that can be very useful aredipolar, zwitterionic com pou nds. Am ino acids have bee nsuggested for that purpose [143], but molecules zwitter-ionic over a wider range of pH are required. The sim-plest one is carboxybe taine, which is zwitterionic, butonly above pH 4, and has been used as a protein solubi-lizer and stabilizer [144]. Mo re prom ising are the non de-tergen t sulfobetaines, whose use in biochemistry wasrecently introduced [145]. These com pou nds have b eenshown to improve solubilization in many biochemicalprocesses, including IEF , while exhibiting no gross dena-turing properties [145, 1461. No te that these com pou ndsinterfere w eakly with acrylamide polym erization, whichcan be overcome by a slight increase in the initiatorsconcentrat ions . The 2-D gel show n in Fig. 5 uses nativeIEF as the first dimension, with a cocktail of TritonX-100, a detergent sulfobetaine (S B 3-14; tetradecyldimethylammoniopropanesulfonate) and a nondetergentsulfobetaine (NDSS B201; pyridiniopropanesulfonate) asthe solubilization mixture. While the result is clearlyinferior to the one obtained with d enatu ring IEF, correctsolubili ty is achieved for m any cellular protein s. In fact,the extraction yield with the native medium describedabove is 50% of the yield obtained with 9 M urea, 4%CH AP S, 40 mM DTT and 20 mM sperm ine. This is animportant step tpwards high resolution, native 2-D elec-trophoresis , which may play a role as a separation toolwhen preservation of the protein activity is necessary forits identification prior to structural analyses.

  • 8/6/2019 Solubilization of Proteins

    15/17

    Elertrophphoresrs 1996, 17 , 813-829

    A

    Solubilization of prote ins 827

    BFigure 5. 2-D electrophoresis of whole cell extracts. Erythroleukemia cells are lysed either in lysis buffer N (1 M SB201, 1% SB 3-14, 0.5% TritonX-100, 0.8% Pharmalytes 3-10, 1mM PM SF, and pepstatin, chymostatin, antipain, aprotinin, leupeptin, 10 pg/mL each) or in lysis buffer D (9 Murea, 4% C HAP S , 20 mM spermine, 40 mM DTT ). The cells are lysed in 10 volumes of lysis buffer per volume of cell pellet, for one h our at 0C(lysis in N) or room tempe rature (lysis in D). The extract is cleared from insolubles (mainly nucleic acids) by ultracentrifugation (200 00 0 g, 1 h) .2-D electrophoresis is carried out with immobilized pH gradients (pH 4-8, linear) for isoelectric focusing [33], rehydrated eith er in 1 M SB201,1% SB 3-14, 0.5% Triton X 100, 0.8% Pharmalytes 3-10 (extract N ) or in 9 M urea, 4% C HAP S , 0.2% Triton X-100, 0.8% Pharmalytes 3-10 an d10 mM DTT (extract D). Second dim ension: 10%T SDS-P AGE . Detection by silver staining. (A) Native lysis and isoelectric focusing (buffer N).(B ) Denatur ing lysis and isoelectric focusing (buffer D). Arrow indicates actin.4 Concluding remarksAs most primary structural analysis methods do notrequire the preservation of three-dimensional conforma-tion, it is often advisable to switch to solubilization andseparation under denaturing conditions as early aspossible in the purification protocol. A few years ago, therequirements of many structural analytic methods wasfar beyond the capacities of electrophoretic methods.Consequently, chromatographic separations were used.These separations often require native conditions, withall the solubility and proteolysis problems describedabove. Thanks to the miniaturization of many structuralanalytic methods, electrophoresis can now be used as amicropreparative tool. This allows the use of new purifi-cation schemes, in which denaturing conditions arewidely used. This in turn has many advantages in termsof solubilization yield, prevention of proteolysis andremoval of interfering compounds, which are among themajor pitfalls in a purification scheme. Therefore, it isoften advantageous not to try to purify the desired pro-tein to homogeneity by standard, native, chromato-graphic methods, but to purify it up to the point where it

    can be reliably identified on a denaturing electrophoreticseparation. The latter will be used, with the desired scale-up if necessary, as the final purification step. Theextreme, but highly recommendable, example of thisapproach lies in the use of high resolution two-dimen-sional electrophoresis as a preparative tool. Conven-tional preparations that make it possible to follow biolo-gical activities sometimes result in only nanogramamounts of the purified protein, with an extremely difi-cult scale-up. It is therefore advisable to use these nano-gram amounts, via two-dimensional gels with silverstaining or radioactive detection, to carry out an identifi-cation of the protein of interest in a more crude extract(e.g. organelle preparation), whose large-scale prepara-tion is much easier. Once this identification has beenmade, two-dimensional electrophoresis is used on thecrude extract at the micropreparative scale to providethe microgram amounts used for structural analysis. Thisprocess using micropreparative two-dimensional electro-phoresis has been greatly facilitated by the use of immo-bilized pH gradients, which have very high loading capa-cities [33, 130, 1471. Such a scheme takes full advantageof the fact that separations under denaturing conditions

  • 8/6/2019 Solubilization of Proteins

    16/17

    828 T. Rabilloud Electrophoresis 1996, 17 , 813-829proceed with a much higher yield and reproducibilitythan those under native conditions. Two-dimensionalelectrophoresis has become the most widely used separa-tion technique to carry out the greatest possible part ofthe solubilization and separation process under dena-turing conditions.

    5 References[ l] Schagger, H., Electrophoresis 1993, 16 , 763-770.[2] Kim, S.Y., Kim, I. G., Chung, S. I., Steinert , P. M., J. Biol. Chem.[3] Reichert, U., Michel, S., Schmidt , R., in : D armon , M. , Blumen-berg, M. (Eds.), Molecular Biology of the Skin, Academic Press,Lo nd on 1993, pp. 107-150.[4] Dill , K. A , , Biochemistry, 1985, 24 , 1501-1509.[5] Tanford, C. , The Hydrophodic Effect, Wiley, New York 1980.[6] Herskovits, T. T. , Jail let , H. , Gad egh eku, B. , J . Bid. Chem. 1970,[7] Manabe , T. , Hayama, E. , O kuyam a, T., Clin. Chem. 1982, 28,[8] Bollag, D. M., Edelstein, S. J. , Protein Methods, Wiley Liss, New[9] Marshall, T., Williams, K. M., Electrophoresis 1992, 13 , 887-888.[lo] Marshall, T. , Abbott , N . J ., Fox, P. , Williams, K. M., Electro-

    [ I l l He l en i us, A , , McCaslin, D. R. , Fries, E. , Tanford, C. , Methods[12] Hjelemeland, L. M ., Methods EnzymoI. 1986, 124, 135-164.[13] Hjelemeland, L. M., Chram bach, A , , Electrophoresis 1981, 2 ,[I41 Neugebau er, J. M ., Methods Enzymol. 1989, 182, 239-252.[15] Navarette, R. : S errano, R. , Biochim. Biophys. Acta 1983, 728,[16] Van Rensw oude, J., Kempf, C. , Methods Enzymol. 1984, 104,1171 Radin, N . S . , Methods Enzymol. 1981, 72, 5-7.1181 Wessel, D., Fliigge, U . I. , Anal. Biochem. 1984, 138, 141-143.[I91 Me nk e, W., Koenig, F., Methods Enzymol. 1980, 69 , 446-452.[20] Penefsky, H. S. , Tzagoloff, A, , Methods Enzymol. 1971, 22 ,[21] Galante, E., Caravaggio, T., Righetti, P. G., Biochirn. Biophys.

    1994, 269, 27979-27986.

    245, 4544-4550.

    824-827.York 1991, pp. 71-93.

    phoresis 1995, 16, 28-31.Enzymol. 1979, 56 , 734-749.

    1-11.

    403-408.329-339.

    204-2 19.Acta 1976, 442, 309-315.Heizm ann, C. W. , Arnold , E. M., Kuenzle , C. C. , J . Bid. Chem.1980, 255, 11504-11511.Smi t h , M. C., Chae , C . B., Biochim. Biophys. Acta 1973, 317,OFarrell , P. H ., J . Biol. Chem. 1975, 250, 4007-4021.Rabilloud, T., Huber t , M . , Tarroux, P., J . Chromatogr. 1986, 351,Zechel, K. , Weber, K. , Eur. J . Biochem. 1977, 77, 133-139.Segers, J ., Rabaey. M. , DeBruyne, G., Bracke, M., Mareel, M.,i n : D u n n , M . J . (Ed. ) , Electrophoresis 86, VCH Weinheim 1986,Glass, W. F., Briggs, R. C., Hnilica, L. S., Science 1981, 211,Shirey, T., Huang, R. C. C., Biochemistry 1969, 8, 4138-4148.Chaudhury, S . , Biochim. Biophys. Acta 1973, 322, 155-165.Sanders , M. M. , Gropp i , V. E. , Browning, E . T. , Anal. Biochem.Willard, K. E ., Gi ome t t i , C . , Ande r son , N . L., OConnor, T. E. ,Anderson, N. G. , Anal. Biochern. 1979, 100, 289-298.Rabilloud, T. , Valette, C. , Lawrence, J. J., Electrophoresis 1994,Mohberg, J ., Rusch, H. P . , Arch. Biochem. Biophys. 1969, 134,Yoshidda, M. , Shimura , K. , Biuchim. Biophys. Acta 1972, 263,Gianazza, E. , Righetti , P. G. , Biochim. Biophys. Acta 1978, 540,

    10-19.

    77-89.

    p p . 642-645.70-72.

    1980, 103, 157-165.

    IS, 1552-1558.577-589.690-695.357-364.

    [37] Adessi, C. , C hapel, A , , Vingon, M., R abilloud, T. , Klein, G . ,Sat re , M. , Gar in , J. , J. Cell Sci. 1995, 108, 3331-3337.[38] Hurkman, W. J . , Tanaka, C. , Plant Physiol. 1986, 81 , 802-806.[39] Gegenheimer , P. , Methods Enzymol. 1990, 182, 174-193.[40] Cremer, F. , Van de Walk, C. , Anal. Biochem. 1985, 147, 22-26.[41] Hari, V., Anal. Biochem. 1981, 113, 332-335.[42] Damerval, C . , De Vienne, D . , Zivy, M., Thiellement, H. , Electro-phoresis 1986, 7, 52-54.Dame rval, C. , Zivy, M., Granier, F. , De Vienne , D. , Adv. Electro-phoresis 1988, 2 , 263-340.Cossu, G. , Pirastru, M. G. , Satta, M., Chiari , M., Chiesa, C. ,Righetti , P. G., J. Chromatogr. 1989, 475, 283-292.Klarskov, K. , Roecklin, D. , Bou cho n, B., Sab atie, J ., Van Dorssa-laer, J. , Bischoff, R., Anal. Biochem. 1994, 216, 127-134.Fritz, J. D., Swartz, D. R. , Greaser, M. L. , Anal. Biochem. 1989,Singh, R. , Biotechniques 1994, 17 , 263-265.Alfageme, C. , Zweidler, A. , Mahowald, A. , Cohen, L. , J . Bid.Chem. 1974, 249, 3729-3736.G u r d , F. R . N . , Methods Enzymol. 1967, 11, 532-541.Riordan, J . F., Vallee, B. L. , Methods Enzymol. 1967, 11,541-548.Griffi th, 0. W., Anal. Biochem. 1980, 106, 207-212.Brune, D. R. , Anal. Biochem. 1992, 207, 285-290.Ruegg, U . T., Riidinger, J. , Methods Enzymol. 1977, 47 , 111-116.Kirley, T. L. , Anal. Biochem. 1989, 180, 231-236.Nor t h , M. J . in : Beynon, R. J. , Bond, J. S. (Eds . ) , ProteolyficEnzymes: A Practical Approach, IRL Press, Oxford 1989,Ballal , N. R. , Goldberg, D. A, , Busch, H. , Biochem. Biophys. Res.C om m . 1975, 62 , 972-982.Colas des Francs, C. , Thiellement, H. , De Vienne, D. , PlantPhysiol. 1985, 78, 178-182.Carter, B . D., Chae, C. B . , Biochemistry 1976, 15, 180-185.Cigarella, C., Negri, G . A, , Guy, O., Eur. J . Biochem. 1975, 53 ,Mayer, J . E. , Hahne, G . , Dalme, K. , Schel l, J. , Plant Cell Rep.Salvesen, G., Nagase, H., in: Beynon, R. J., Bond, J . S. (Eds.),Protealyric Enzymes: A Practical Approach, IR L Press, OxfordGranzier, H. L. M., Wang, K. , Electrophoresis 1993, 14, 56-64.Cleveland, D. W., Fischer, S. G., Kirschner, M. W., Laemmli,U . K ., J . Biol. Chem. 1977, 252, 1102-1106.Harrison, P. A., Black, C. C., Plant Physiol. 1982, 70 , 1359-1366.Wu. F. S.. Wane. M. Y.. Anal. Biochem. 1984. 139. 100-103.

    180, 205-210.

    pp. 105-124.

    457-463.1987, 6, 77-81.

    1989, pp. 83-104.

    I , LI I ,[66] Gianazza, E. , Righetti , P. G. , J. Chromatogr. 1980, 193, 1-8.1671 Reynolds, J. A, , Tanford, C. , Proc. Natl. Acad. Sci . USA 1970, 66 ,[68] Reynolds, J. A , , Tanford, C. , J . Bid. Chem. 1970 ,245, 5161-5165.[69] Weber, K. , Osb orn, M ., J. Bid. Chem. 1969, 244, 4406-4412.[70] Lopez, M. F., Pat ton, W. F., Utterback, B. F., Chung-Welch, N. ,Barry, P., Skea, W. M., Cambria, R. P., Anal. Biochem. 1991, 199,35-44.

    1002-1007.

    [71] Laemmli , U. K. , Nature 1970, 227, 680-685.[72] Wilson, D., Hall, M. E., St one , G. C., Rubin, R. W., Anal. Bio-1731 Panyim, S . , Chalkley, R., J. Bid. Chem. 1971, 246, 7557-7560.[74] Amory, A. , Foury, F., Goffeau, A, , J. Biol. Chem. 1980, 255,[ 75 ] M ocz , G . , B a h t , M . , Anal. Biochem. 1984, 143, 283-292.[76] MacFarlane, D., Anal. Biochem. 1983, 132, 231-235.[77] Caglio, S . , Righetti, P. G., Electrophoresis 1993, 14 , 554-558.[78] Eley, M. H., Burns, P. C., Kannapell, C. C., Campbell , P. S. , Anal.1791 De shp an de , V. V., Bod he, A. M ., Pawar, H. S. and Vartak, H. G. ,[80] Chiari , M., Manzocchi, A , , Righetti , P. G ., J. Chromatogr. 1990,[81] Geisthardt, D. , Kruppa, J. , Anal. Biochem. 1987, 160, 184-191.[82] Bonaventura, C. , Bonaventura, J. , Stevens, R. , Millington, D. ,[83] Akins, R. E. , Lev in, P. M., Tuan, R. S., Anal. Biochem. 1992, 202,

    chem. 1977, 83 , 33-44.9353-9357.

    Biochem. 1979, 92, 41-419.Anal. Biochem. 1986, 153, 227-229.500, 697-704.

    Anal. Biochem. 1994, 222, 44-48.172-178.

  • 8/6/2019 Solubilization of Proteins

    17/17

    Electrophoresis 1996, 17 , 813-829 Solubilization of pro te ins 829[84] Moos, M ., Nguyen, N. Y., Liu, T. Y., J. Bid. Chem. 1988, 263,[8 5 ] Lyubim ova, T. , Caglio, S., Gelfi , C. , Righetti , P. G. , Rab illoud, T. ,[86] Chiari , M . , Righetti , P. G., Electrophoresis 1995, 16, 1815-1829.[87] Caglio, S . , Righetti, P. G., Electrophoresis 1993, 14 , 997-1003.[88] Caglio, S. , Righetti , P. G. , Electrophoresis 1994, 15, 209-214.[89] Rabilloud, T. , Girardot, V. , Lawrence, J. J. , Electrophoresis 1996,[90] MacFarlane, D. , Anal. Biochem. 1989, 176, 457-463.[91] Chae, H. Z. , Uhm , T. B., Rhee, S. G . , Proc. Natl. Acad. Sci. USA[92] Paulson, J. R. , Mesner, P. W., Delrow, J. J. , M ahmoud , N . N . and[93] Dewald, B. , Dulaney, J. J., Touster, O., Methods Enzymol. 1974,[94] Newby, A. C. , Chrambach, A, , Biochem. J. 1979, 177, 623-630.[95] Schagger, H. , von Jagow, G. , Anal. Biochem. 1991, 199, 223-231.[96] Koide, M. , Fukuda, M. , Ohbu, K ., Watanabe, Y. , Hayashi , Y.,[97] Hisabori, T. , Inoue, K. , Akabane, Y. , Iwakami, S. , Manabe, K. ,

    6005-6008.Electrophoresis 1993, 14, 40-50.

    17> 67-73.

    1994, 91, 7022-7026.Cieselski, W. A,, Anal. Biochem. 1992, 203, 227-234.32 , 82-91.

    Takagi, T., Anal. Biochem. 1987, 164, 150-155.J. Biochem. Biovhvs. Methods 1991. 22. 253-260._ I /[98] Schagger, H., Cram er, W. A,, von Jagow, G., Anal. Biochem. 1994,21 7, 220-230.

    [99] Von Jagow, G. , Schagger, H. , A Practical Guide to Membrane[loo] Wall, J . S. , Fey, D. A., Paulis, J. W., Cereal Chem. 1984, 61 ,[I011 Branlard, G . , Dardevet, M. , Cereal Sci. 1985, 3, 329-343.[lo21 Vecchio, G. , Righetti , P. G. , Zanoni, M., Artoni, G. , Gianazza,E ., Anal. Biochem. 1984, 137, 410-419.[lo31 Artoni, G. , Gianazza, E., Zanon i , M ., Gelfi , C. , Tanzi, M. C. ,Barozzi, C. , Ferruti , P., Righetti , P. G . , Anal. Biochem. 1984, 137,[lo41 Zewert, T. , Harrington, M., Electrophoresis 1992, 13 , 817-824.[IOS] Zewer t , T., Harrington, M ., E/ectrophoresis 1992, 13 , 824-831.[lo61 Siemankowski, R. F. , Giambalvo, A, , Dreizen, P. , Physiol. Chem.[lo71 T uszynski, G. P. , Buck, C. A , , Warren, L. , Anal. Biochem. 1979, 93,[lo81 Nakam ura, K . , Okuy a, Y., K atahira, M ., Yoshida, S. , Wada, S. ,Okuno, M. , J. Biochem. Biophys. Methods 1992, k24, 195-203.[lo91 Lin, W., Kasamatsu, H. , Anal. Biochem. 1983, 128, 302-311.[110] M atthaei, S . , Baly, D. L., Horuk, R. , Anal. Biochem. 1986, 157,[ l l l ] R e dd y, S. G., Cochran, B. J. , Worth, L. L. , Knutson, V. P.,Haddox, M. K. , Anal. Biochem. 1994, 218, 410-419.[112] Bonfils, C., Daujat, M., Chevron, M. P., Dalet-Beluche, I., Anal.

    Biochem. 1994, 218, 80-86.[113] Ame s, G . F. L. , Nikaido, K. , Biochemistry 1976, 15 , 616-623.[I141 Webe r, K., Kuter, D. J. , J. Biol. Chem. 1971, 246, 4504-4509.[115] Remy, R., Amb ard-Bretteville, F., Methods Enzymol. 1987, 148,[116] March, J., Advanced Organic Chemistry, McGraw-Hi l l , London[117] Dunn, M. J. , Burghes , A. H . M., Electrophoresis 1983, 4, 97-116.[I181 Gianazza , E. , Rabilloud, T., Quaglia, L., Caccia, P., AStrua-Testori, S. , Osio, L. , Grazioli , G. , R ighetti , P. G. , Anal. Biochem.

    Purification, Academic Press, San Diego 1994.141-146.

    420-428.

    Phys. 1978, 10, 415-434.329-338.

    123-128.

    623-632.1977, pp. 83-84.

    1987, 165, 247-257.

    [119] Rab illoud, T. , Gianazza, E., Cat t o , N., Righetti, P. G., Anal. Bio-[120] Holloway, P. J., Arundel, P. H., Anal. Biochem. 1988, 172, 8-15.[121] Witzman n, F., Jarnot, B. , Parker, D. , Electrophoresis 1991, 12,[122] Clare Mills, E. N ., Freedm an, R. B., Biochim. Biophys. Acta 1983,[I231 Satta, D. , Schapira, G. , Chafey, P. , Righetti , P. G. , Wahrmann,11241 Gyenes, T., Gyenes, E., Anal. Biochem. 1987, 165, 155-160.[125] Perdew, G . H. , Schaup, H. W., Selivonchick, D. P., Anal. Biochem.1983, 135, 453-455.[126] Bossi, A, , Righetti , P. G. , Riva, E. , Zerilli, L., Electrophoresis1996, 17 , in press.[127] Hochstrasser, D. F., Harrington, M. G. , Hochstrasser, A. C. ,Miller, M. J. , Merril, C. R. , Anal. Biochem. 1988, 173, 424-435.[128] Hagel, P., Gerding, J. J . T. , Fieggen, W., Bloemendal, H. ,

    Biochim. Biophys. Acta 1971, 243, 366-373.[129] Gerding , J. J. T., Koppers, A. , Hagel, P., Bloemendal, H. ,Biochim. Biophys. Acta 1971, 243, 374-379.[130] Bjellqvist, B., Sanchez, J. C., Pasquali, C., Ravier, F., Paquet, N. ,Frutiger, S., Hugh es , G. J . , Hochst rasser , D. F . , Electrophoresis

    [131] Burghes, A. H. M., Patel , K. , Dunn, M. J . , in: Neuhoff, V. (Ed.)[132] Steinfeld, R. C. , Vidaver, G. A, , Biophys. J . 1981, 33 , 185.[133] Al thaus , H . H . , Kloppner , S . , Poehling, H. M., Neuhoff, V. ,[134] Herskovits, T. T.,