Sodium dodecyl sulfate polyacry-lamide gel electrophoresis (SDS-PAGE) is the most widely used ana-lytical method to resolve separate

components of a protein mixture. It isalmost obligatory to assess the purity of aprotein through an electrophoretic method.SDS-PAGE simultaneously exploits differ-ences in molecular size to resolve proteinsdiffering by as little as 1% in their elec-trophoretic mobility through the gel matrix(1). The technique is also a powerful tool forestimating the molecular weights of proteins(2, 3). The success of SDS-PAGE as an in-dispensable tool in protein analysis has beenattributed to three innovations that permit-ted the correlation of electrophoretic mobil-ity with a protein’s molecular mass (4). Firstwas the introduction of discontinuous buffersystems where the sample and gel runningbuffers differ in both composition, Tris-HCl/Tris-glycine, and pH, 6.8/8.3, respec-tively (5, 6). Discontinuous buffer systemsallow larger sample volumes to be loadedwhile maintaining good resolution of sample

components because the proteins are fo-cused, or “stacked,” as thin bands prior toentering the resolving gel. Second was theuse of the detergent sodium dodecyl sulfate(SDS) and reducing agents to denature pro-teins (7). SDS binds strongly to proteins atan approximate ratio of 1 dodecyl sulfatemolecule per 2 amino acid residues (8).Therefore, the negative charge/unit massratio when SDS is bound to the polypeptidechain is similar for all proteins. Third wasthe combination of the first two discoveriesemploying a simple Tris-glycine buffersystem (9). More recently, buffer combina-tions such as Tris-borate (10) and Tris-tricine (11) have improved the resolvingpower of the original methods. ModernSDS-PAGE has evolved to use microslabprecast gels (12). Precast and packaged gelsin a wide variety of gel formulations, acry-lamide percentages, thicknesses, well for-mats, and buffer systems are now commer-cially available from several manufacturers.Therefore, successful SDS-PAGE analysis ofprotein samples no longer depends on te-

dious gel casting, buffer preparation and ap-paratus set-up, but on careful sample prepa-ration and treatment prior to loading thegel. This article describes techniques andprocedures as a guide for preparation of pro-tein samples for SDS-PAGE analysis.

Sample buffer preparation

To ensure consistent and successfulPAGE analysis, the highest purity reagentsshould be used to prepare sample bufferstock solutions. After a reliable source ofelectrophoresis reagents has been identified,the vendor and buffer component chemicalsshould be maintained. High purity elec-trophoresis, Ultrol® grade, and molecular bi-ology grade reagents are available throughNovagen’s partner brand, Calbiochem.Solutions must be carefully and safely pre-pared, dated, and chemical lot numbersrecorded. Concentrated stock solutionsshould not be stored for long periods oftime. Tris base, rather than Tris-Cl, shouldbe used for buffer preparation and pH ad-justment made with HCl. Use of Tris-Cl

10inNovations 13

ARTICLE

Preparation of protein samples for SDS-polyacrylamide gelelectrophoresis: procedures and tipsAnthony C. Grabski1 and Richard R. Burgess2—1Novagen, Inc. and 2McArdle Laboratory for Cancer Research, University ofWisconsin-Madison, Madison, WI 53706

continued from page 9

pensive method to maintain very low basalexpression levels of T7 RNA polymerase inthe λDE3 lysogenic expression hosts usedin the pET System. This is especially truewhen λDE3 hosts carrying pET plasmidsare grown to stationary phase. A disadvan-tage with glucose addition is that after aninitial rapid growth phase the metabolicbreakdown products of glucose will lead toacidic culture conditions and lower celldensity at stationary phase. The data pre-sented in Figure 2 demonstrate that stronginduction can be achieved from λDE3 lyso-gens in the presence of glucose for sometarget proteins. Note, however, that theoret-ically the strongest induction of T7 RNApolymerase would be expected when glu-cose is absent and cAMP levels are elevated.Accordingly, in some cases (see precedingarticle), higher target protein expression

may be observed in the absence of glucose.Overall, the optimal combination of strin-gent uninduced repression and high in-duced expression may be achieved by initialgrowth in the presence of glucose, followedby switching to medium without glucosefor induction.

Novagen’s recommendations for growthand induction of pET constructs in expres-sion hosts are based on the information pre-sented above. For innocuous proteins, anypET vector and λDE3 lysogen are suitablein a variety of media. But for proteins thatare potentially toxic to the bacterial cell, werecommend using either a pET vector witha T7lac promoter or expression hosts thatcarry the pLysS plasmid. In addition, ourgeneral advice is to avoid growing a λDE3lysogen carrying a pET plasmid to station-ary phase. If the cells must be grown to sta-

tionary phase, we recommend the additionof 0.5 to 1.0% glucose to the medium, sothat the glucose effect can be exploited toreduce basal expression.

REFERENCES1. Grossman, T. H., Kawasaki, E. S.,

Punreddy, S. R., and Osburne, M. S.(1998) Gene 209, 95–103

2. Pan, S. and Malcolm, B. A. (2000)BioTechniques 29, 1234–1238.

3. Le Grice, S. F. J., Matzura, H., Marcoli,R., Iida, S., and Bickle, T. A. (1982) J.Bacteriol. 150, 312–318.

4. Kelley, K. C., Huestis, K. J., Austen, D.A., Sanderson, C. T., Donoghue, M.A., Stickel, S. K., Kawasaki, E. S., andOsburne, M. S. (1995) Gene 15,33–36.

11inNovations 13

will result in a higher ionic strength, poormigration and diffuse protein bands (13). Arespirator or dust mask should be wornwhen handling powdered SDS. Sulfhydrylreagents, dithiothreitol (DTT) and 2-mer-captoethanol (2-ME) can be unstable in so-lution and are toxic. These chemicals shouldbe measured in a fume hood while wearinggloves and safety glasses. Although 2-ME ishistorically the chemical of choice for reduc-tion of protein disulfide bonds in SDS-PAGE (7, 9), DTT is also a very effective al-ternative (14). Glycerol is added to increasethe sample density, facilitating gel loadingand preventing convective migration out ofthe sample wells. A small amount ofbromphenol blue is added as a visual aidduring sample loading and as a tracking dye,allowing easy monitoring of electrophoreticprogress.

The sample buffer recipes listed in Table1 are commonly used for Tris-glycine SDS-PAGE analysis of protein samples under de-naturing, reduced conditions (7, 9, 13).When preparing these buffers, wear glovesto avoid keratin (skin protein) contamina-tion. A heterogeneous cluster of bandsaround 55 kDa can be seen when a keratin-contaminated sample or sample buffer isused. This is particularly obvious with highsensitivity silver staining methods. Thesample buffer should be divided into 1-mlaliquots and can be stored frozen (–70°C)for several months. Prior to use, warm(37°C) and mix the solution briefly to com-pletely dissolve the SDS.

Protein sample preparation

Sample preparation is critical for clearand accurate resolution of protein bands.Photographic quality results are routinelypossible if samples are carefully prepared.Common mistakes during sample prepara-tion include using an incorrect protein-to-sample buffer ratio, delayed heating, over-heating, failure to remove insolublematerial, and overloading and underloadingof protein. To prevent inadequate samplebuffer-to-protein ratios, overloading, andunderloading of samples, the protein con-centration of the sample should be deter-mined using a standard protein assay such asthe CB-Protein Assay™, Non-InterferingProtein Assay™, or bicinchoninic acid

(BCA) assay prior to sample buffer addition.Loading too much protein will result in dis-torted, poorly resolved bands in the over-loaded lane and distorted electrophoreticpatterns in adjacent lanes. Underloadingsimply prevents detection of minor compo-nents while even major bands will be toofaint for photographic reproduction of the

gel. Depending on the well size and gelthickness, the amount of protein loadedshould range from 0.5–4.0 µg for purifiedsamples and from 40–60 µg for crude sam-ples if a Coomassie blue stain (e.g.,RAPIDstain™) is used. Silver stainingmethods (such as the FASTsilver™ Kit) areapproximately 100-fold more sensitive, andtherefore require less protein per sample.

SDS-PAGE sample buffer treatment isdesigned to completely dissociate all pro-teins into their subunit polypeptides.Proteins heated in the presence of SDS aredenatured and imparted with a strong nega-tive charge. Thiol reagents in the samplebuffer reduce disulfide bonds. It is impor-tant to use enough sample buffer in order tomaintain an excess of SDS. Most polypep-tides bind SDS in a constant mass ratio of

1.4 µg SDS per 1.0 µg polypeptide, but aratio of 3:1 is recommended (15). The 2Xsample buffer prepared as shown in Table 1contains 40 µg/µl SDS. Maintained reduc-tion of protein sulfhydryls is essential inorder to prevent intramolecular disulfidebond formation through oxidized cysteines.If artifactual band heterogeneity or unusualdoublets are noted in SDS-PAGE resultsfrom samples containing sulfhydryls, insuf-ficient reducing agent was present duringsample treatment or the oxidation of cys-teines may have occurred during the stack-ing phase of electrophoresis. These artifactsmay be prevented if samples are treated withiodoacetamide (IAA) after heating in the ap-propriate concentration of sample buffer.The IAA treatment irreversibly blockssulfhydryls and destroys excess reducingagent (16). Therefore, the sample bufferrecipes in Table 1 do not necessarily indicatefunctional dilution factors, but rather theyare convenient stock concentrations permit-ting correct addition of reagents to samplesof high and low protein concentration.

Delayed heating of samples after samplebuffer addition or excessive heating cancause electrophoretic artifacts due to proteindegradation and peptide bond cleavage, re-spectively. Upon addition of SDS samplebuffer, samples should be immediatelymixed and heated to 85°C for three min-utes. This treatment is usually sufficient toreduce disulfides, solubilize and dissociateproteins without peptide bond cleavage.Addition of SDS sample buffer will begin todenature most proteins. However, proteasesare known to be resistant to SDS denatura-tion alone (15, 17). Partially denatured sam-ples (particularly crude extracts) are there-fore extremely sensitive to proteolyticdegradation as protease active sites withinthe polypeptides become exposed by SDStreatment. Immediate heating limits degra-dation by completely denaturing all proteinsincluding resistant proteases through thecombination of heat, SDS, and reductant.Protease inhibitors may also be used duringsample preparation to limit proteolysis.Excessive heating, e.g., 100°C for prolongedperiods, may break peptide bonds or causeselective aggregation and band smearing(18). Asp-Pro bonds have been demon-strated to be sensitive to thermal cleavage.

Sample preparation is critical for clear and accurate resolution

of protein bands.

Table 1. SDS-PAGE sample buffer recipes

Component Concentration

2X 4X

Tris-HCl, pH 6.81 0.125 M 0.25 M

SDS 4% 8%

2-ME2 5% 10%

DTT3 0.15 M 0.3 M

Glycerol 20% 30%

Bromphenol blue .01% .02%

1. Prepared using Tris base, pH adjusted with HCl.

2. If 2-ME is used, omit DTT.

3. If DTT is used, omit 2-ME.

continued on page 12

In some cases more extreme heating may benecessary to completely denature the protein(19, 20). Therefore, if prolonged heating at100°C is necessary for complete dissociationof a thermally stable protein, the effects ofsuch treatment upon peptide bond cleavagemust be considered (21). Some proteinssuch as histones and membrane proteinsmay not completely dissolve by heating inSDS sample buffer alone and may requireaddition of 6–8 M urea or a nonionic deter-gent such as Triton X-100 (22, 23). Afterheat treatment in SDS sample buffer, insolu-ble material must be removed by brief cen-trifugation. This is easily accomplished by atwo-minute spin in a microcentrifuge at17,000 × g. Failure to remove precipitatedinsoluble material from the sample willcause streaking within the gel. The super-natant of the treated sample is now ready toload. The sample may be stored at 4°Covernight or frozen at –20°C for longer peri-ods. Warm stored samples briefly at 37°C toredissolve the SDS and recentrifuge toremove insoluble material prior to loading.

Preparation of difficult samples

Samples that are dilute, acidic, very vis-cous, or that contain interfering compoundspose unique challenges to the SDS-PAGEanalysis method. However, these difficultsamples can be analyzed by SDS-PAGEthrough the application of one or more ofthe following pre-treatment techniques.Samples too dilute for analysis can be con-centrated by several methods includinglyophilization, spin concentrators, dialysisagainst concentrated polyethylene glycol(PEG), and absorption of excess solvent byexposure of the dialysis bag containingsample to dry PEG, Aquacide or gel filtra-tion media such as Sephadex®. Samples con-centrated through these methods may be di-alyzed against 50 mM Tris-HCl, pH 6.8 toremove low molecular weight impuritiesprior to addition of SDS sample buffer.Dilute samples, acidic samples and samplescontaining interfering compounds such aspotassium, guanidine hydrochloride, orionic detergents can be precipitated bytrichloroacetic acid or acetone to concen-trate the proteins and remove contaminants.Protocols for TCA, acetone/methanol, andethanol precipitation are described in refer-ences 4, 15, 17 and 24. A modified acetone

precipitation method is also described inNovagen Technical Bulletin 012, available atwww.novagen.com. Crude cell extracts areoften extremely viscous due to the high con-centration of unsheared nucleic acids. Thehigh viscosity is problematic during gelloading, because samples are difficult topipet and will not be evenly distributed inthe sample well. Viscosity can be eliminatedby treatment of samples with Benzonase®

Nuclease prior to addition of sample buffer.This recombinant endonuclease completelydegrades all forms of DNA and RNA and isfree from proteolytic activity. Viscosity canalso be reduced by physical shearing of thenucleic acids through sonication or by vigor-ous vortex mixing of the heated sample.Employing these pre-treatment protocolswill allow successful SDS-PAGE analysis ofsamples that are very dilute, viscous or cont-aminated with interfering compounds.

REFERENCES1. Scopes, R. K. (1994) “Protein

Purification: Principles and Practice,”3rd ed., Springer Verlag, New York.

2. Weber, K., Pringle, J. R., and Osborn,M. (1971) Meth. Enzymol. 26, 3–27.

3. Chambach, A. and Rodbard, D. (1971)Science 172, 440–450.

4. Marshak, D. R., Kadonaga, J. T.,Burgess, R. R., Knuth, M. W., Brennan,W. A., and Lin, S. -H. (1996)“Strategies for Protein Purification andCharacterization,” Cold Spring HarborLaboratory Press, New York.

5. Ornstein, L. (1964) Ann. NY Acad. Sci.121, 321–349.

6. Davis, B. J. (1964) Ann. NY Acad. Sci.121, 404–427.

7. Shapiro, A. L. and Maizel, J. V. (1969)Anal. Biochem. 29, 505–514.

8. Reynolds, J. A. and Tanford, C. (1970)Proc. Natl. Acad. Sci. USA 66,1002–1007.

9. Laemmli, U. K. (1970) Nature 227,680–685.

10. Neville, D. M. and Glossmann, H.(1974) Meth. Enzymol. 56, 58–66.

11. Schagger, H. and Von Jagow, G. (1987)Anal. Biochem. 166, 368–379.

12. Matsudaira, P. T. and Burgess, R. R.(1978) Anal. Biochem. 87, 386–396.

13. Patel, D. (1994) in “GelElectrophoresis,” Rickwood, D. andHames, B. D., eds, pp. 3–5, John Wiley& Sons, New York.

14. Cleland, W. W. (1964) Biochemistry 3,480–482.

15. Hames, B. D. (1990) in “GelElectrophoresis of Proteins,” Hames, B.D. and Rickwood, D., eds., pp. 1–147,Oxford University Press, New York.

16. Crow, M. K., Karasavvas, N., and Sarris,A. H. (2001) BioTechniques 30,311–316.

17. Gallagher, S. R. (2000) in “CurrentProtocols in Protein Science,” Coligan,J. E., Dunn, B. M., Ploegh, H. L.,Speicher, D. W., and Wingfield, P. T.,eds., pp. 10.1.29–10.1.34, John Wiley& Sons, New York.

18. Gallagher, S. R. and Leonard, R. T.(1987) Plant Physiol. 87, 120–125.

19. Bensadoun, A., Ehnholm, C., Steinberg,D., and Brown, W. V. (1974) J. Biol.Chem. 249, 2220–2227.

20. Slutzky, G. M. and Ji, T. H. (1974)Biochim. Biophys. Acta 373, 337–346.

21. Deutsch, D. G. (1976) Anal. Biochem.71, 300-303.

22. Franklin, S. G. and Zweidler, A. (1977)Nature 266, 273–275.

23. Siu, C. H., Lerner, R. A., and Loomis,W. F. (1977) J. Mol. Biol. 116,469–488.

24. Hager , D. A. and Burgess, R. R. (1980)Anal. Biochem. 109, 76–86.

12inNovations 13

ARTICLEcontinued from page 11

Product Size Cat. No.

Benzonase® Nuclease,Purity > 90% 10,000 U 70746-3

Aquacide I 1 kg 1785

Aquacide II 1 kg 17851

Aquacide III 1 kg 17852

CB-Protein Assay™ 1 kit 219468

Non-InterferingProtein Assay™ 1 kit 488250

Tris Base, Ultrol® 100 g 648311Grade 1 kg

Cleland’s Reagent, 1 g 233155Reduced (DTT) 5 g

Glycerol, Molecular 100 ml 356352Biology Grade 1 liter

Sodium n-Dodecyl Sulfate,High Purity 25 g 428016

4X SDS Sample Buffer 2 ml 70607-3

Perfect Protein™Markers, 15–150 kDa 100 lanes 69149-3

Perfect Protein™Markers, 10–225 kDa 100 lanes 69079-3

Trail Mix™ ProteinMarkers 100 lanes 70980-3

RAPIDstain™ 1000 ml 553215

FASTsilver™ Kit 1 kit 341298

= Calbiochem brand product