Weber K. y Osborn M. 1969. the Reliability of Molecular Weight Determinations by Dodecyl

7/28/2019 Weber K. y Osborn M. 1969. the Reliability of Molecular Weight Determinations by Dodecyl

1/7


2/7

Issue of -4ugust 25, 1969 K. W&r and M. Osborn 4407ylase, tropomyosin, and paramyosin was performed using iodo-acetamide in sodium dodecyl sulfate or 8 sx guanidine-IICl (6).lhe follo\ving procedure is based on the original descriptionsby Shapiro et al. (I), by Ornstein (i), and Davis (8).

Preparation 0J Protein SolutionsThe lxotcins were incubated at 37 for 2 hours in 0.01 M sodiumphosphate buf fer, $1 7.0, 1 y0 in SDS, and 1% in ,8mercapto-

ethanol. Tropomyosin, paranlyosin, and myosin were dis-sol\-cd in this buf fer in the prcsencc of 8 M urea. The proteinconcentration ~7~s nornlall~- betlvccn 0.2 and 0.6 mg l)cr ml.&1fter incubation the protc,in solutions IT-ere dialyzed for ,xvera lhours at room temperature against 500 ml of 0.01 M sodiumphosphntc buf fer, $1 7.0, containing 0.1% SDS and 0.1%P-merc:il~toc:tb:I~lol . In most casts the dialysis step may beomitted and the protein dissolved d irectly in dialysis buffer.

Preparation of GelsGel buffer contained 7.8 g iY:rI-I210,,.H20, 38.6 g of Ns~IIlO~~

7H20, 2 g of SDS per liter. For the lO$G acrylamide solution,22.2 g of acrylamide and 0.6 g of r~~ethylcnebisncrylamide weredissolved in water t ,o give 100 ml of solution. Insoluble materialwas removed by filtrat ion through Whatmun No. 1 filter paper.The solution was kept nt 4 in a dark bottle. Gels with in-creased and decreased cross-linker contained twice and hal f theconcentration of cross-linker, respccti rcly.

The glass gcll tubes were 10 cm long with an inner diameter of6 111111. Before use the)- were soaked in cleaning solution, rinsed,and oreli-dried. For a typica l run of 12 gels, 15 ml of gel buf ferwere deacrated and rnised with 13.5 ml of acrylamide solution.After further deaeration, 1.5 ml of freshly made ammoniumpersulfate solution (15 mg per ml) and 0.045 1111 f N, N ,N ,h-tetrameth?-lethJ-leaecliamille were added. After mixing, eachtube ~\-as filled with 2 ml of the solution. Before the gel hard-ened a few drops of water were layered on top of the gel solution.After 10 to 20 min an interface could be seen indicating that thegel hat1 solidified. Gels n-ith normal amount of cross-linkerremain clear, those with doubled cross-linker turn opaque. *Justbefore use t,he mater layer was sucked of f, and the tubes wereplaced in the clectrophoresis apparatus.

Preparation of Samp lesFor each gel, 3 ~1 of tracking dye (O.O5c;/, 1)romphenol blue in

\I-ater), 1 drop of glycerol, 5 ~1 of Incrcaptoeth:uiiol, and 50 ~1 ofdialysis buf fer ~7-a~ nlisecl in a sn~rll test tube. Then 10 to 50~1 of the protein solution were added. After mixing, the solu-tions were applied on the gels. Gel buff er, diluted 1 :I withI\-atcr, K:IS carefully layered on top of each sample to fil l t,hetubes. The tTx-o compartments of the electrophoresis apparatusx7-erc filled with gel buffer, diluted 1:l with water. Electro-phoresis XI performed at a constant current of 8 ma per gelwith the positive electrode in the loTIer chamber. Under theseconditions the marker dye morcd three-quarters through thegel in approximately 4 hours. The time taken to run the gelmay be decreased by decreasing t,he molarity of the gel buffer.2After elect-rophorcsis, the gels xxrc removed from the tubesby squirting lvater from n syringe b&J\-een gel and glass wall andby using a pipette bulb to exert pressure. The length of the geland the distance moved by the dye were measured.

2 A. Burgess, personal communication.

Staining and &staining-The gels were placed in small tubesfilled with st,aining solution prepared by dissolving 1.25 g ofCoomassie brilliant blue in a mixture of 454 ml of 50 c/ methanoland 46 ml of glacial acetic acid, and removing insoluble materialby filtrat ion through Whatman Ko. 1 filter paper. Staining wasat room teml)erature. The time varied frorn 2 to 10 hours. Thegels were removed from the st,aining solution, rinsed with distilledwater, and placed in destaining solution (75 ml of acetic acid, 50ml of methanol, and 875 ml of water) for a minimurn of 30 min.The gels were then further destained electrophoretically for 2hours in a gel clectrophoresis apparatus using destaining solution.The length of the gels after destaining and the positions of theblue protein zones T\-cre recorded. The gels were stored in 7.5y0acetic acid solution.

The gels swell some 5% in t,he acidic solution used for stainingand destaining. Gels with lower amount of cross-l inker showmore swelling. Therefore the calculation of the mobili t,y has toinclude the length of the gel before and after staining as well asthe mobility of the protein and of the marker dye. Assumingeven sxelling of the gels, the mobility was calculated asMobility = distance o f protein migrationlength after destaining

x length before stainingdistance of dye migrationThe mobili ties were plotted against the known molecular weightsexpressed on a semi-logarithmic scale.

Time Required for Molecular Weight Determination- It ispossible to obtain the molecular weight of a particular proteinwithin a day: preparation of gels and samples, 2 hours; elcctro-phoresis, 3 to 4 hours; removing the gels, + hour; staining, 2hours; destaining, 2 to 3 hours.

Amount 0J ProteinUsually 0.01 mg of protein was applied per gel. The amountcould be lon-ered if the gel was stained for a longer period. I f0.1 mg was applied although the trailing edge of the band \vasdif fuse , the leading edge IT-as still very sharp and molecular

weights could still be found very accurate ly.E&ion of Proteins jrom Gels

On each of two gels, 0.1 mg or more was run. One gel wasst.ored wrapped in Saran 1Vrap at 4. The other gel was stainedand destained to localize the protein band. The correspondingvolume of the first gel was cut inlo smaller pieces. The gelmaterial was suspended in a small amount of 0.1 o/o SDS solutionand kept for several hours at 37. The solution was withdrawnand a second elution was performed. The combined cluentswere lyophilized in a conical centrifuge tube. Distilled materwas added to obtain a 1 y0 SDS solution (usually about 50 to 100~1). Then nine parts o f ice-cold acetone were added for onepart of solution. The protein precipitated and could be cen-trifuged of f, whereas the detergent stayed in solution. Theprecipitate could be transferred to a hydro lysis tube by dissolvingit in 5 7. piperidine solution or in 12 N HCl.

RESULTSTwo procedures were used to examine the relationship be-

tween electrophoretic mobilities and molecular weights of variousproteins. Either several SDS-denatured proteins were mixed

bygu

est,onMay7,2012

www.jbc.org

Downloadedfrom
http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/http://www.jbc.org/


3/7

4408 Molecular Weights by Gel Ebctrophoresis Vol. 244, No. 16and run together on one gel or the proteins were run in a parallelmanner on different gels. Identical results were obt.ained byboth methods.

Fig. 1, A, B, and C, shows examples of the separation of severaldifferent polypeptide chains on a single gel. Remarkably goodseparation is obtained for the polypeptide chains of cat.alase,fumarase, aldolase, glyceraldehyde phosphate dehydrogenase,carbonic anhydrase, and myoglobin (Fig. L/l). The identity ofeach band was established by running each protein on a dif ferentgel. In each case a single protein band with an electrophoreticmobility corresponding to the one assigned to it in the mixturewas observed. Fig. 1B shows a similar mixture which was runon a diffe rent occasion, but without myoglobiu. In Fig. IC t.hesame proteins were run as in Fig. IA, but substituting liveralcohol dehydrogenase for aldolase. Again, separation of allsix polypeptide chains was observed. The control gel for liveralcohol dehydrogenase indicated only one band with a mobilit?corresponding to that assigned to it in the mixture. The molec-ular weights for the different polypeptide chains are taken fromTable I, in which the proteins we studied by SDS gel electro-phoresis are listed. The pictures illustrate the dependence ofthe mobility on the logarithm of the molecular weight of thepolypeptide chains (1). The separation in the molecular weightrange of 30,000 to 60,000 is excellent. It can be improvedfurther either by increasing the length of the gel or by decreasingthe amount of protein applied.

As an example of the method in which each protein is run on adifferent gel, Fig. 2 illustrates the determination of the molecular

FIG. 1. Separation of the polypeptide chains of diffe rentproteins in gels with the normal amount of cross-linker. Theproteins are listed from lop to bottom, and the molecular weightsgiven for the different polypeptide chains are taken from Table I.A, catalase (60,000), fumarase (49,000), aldolase (40,000), glycer-aldehyde phosphate dehydrogenase (36,000), carbonic anhydrase(29,000), and myoglobin (17,200); B, same as A, but omitting themyoglobin. This gel was run on a different occasion; C, catalase,fumarase, liver alcoho l dehydrogenase (41,000), glyceraldehydephosphate dehydrogenase, carbonic anhydrase, and myoglobin.

TABLE IProteins studied bv SDS-electrophoresis

The table lists molecular weights of the polypeptide chain staken from the literature. Proteins which under native condi-tions exist as oligomers are indicated by an asterisk.

ProteinMyosin*. ....................p-Galactosidase*. ............Paramyosin .................Phosphorylase a*. ..........Serum albumin. .............L-Amino acid oxidase. ......Cat,alase*, ..................Pyruvate kinas e*. ........ :Glutamate dehydrogenase*. ..Leucine amino peptidase .....r-Globulin, H chain*. .......Fumarase*. ..................&albumin. ................Alcoh ol dehydrogenase (liver)Enolase* ....................Aldol ase*. ...................Creat,ine kinase*. ............D-Amino acid oxidase*. ......Alcohol dehydrogenase

(yeast) * .................Glyceraldehyde phosphatedehgdrogenase*. ...........Tropomyosin* ..............

Lactat,e dehydrogenase*. .....Pepsin .......................Aspartate transcarbamylase,

C cha in*. .................Carboxy peptidase A. ........Carbonic anhydrase ..........Subt.ilisin. ..................r-Globulin, L chain.*. .......Chymotrypsinogen. .........Trypsin ....................Papain (carboxymethyl) .....p-Lactoglobulin*. ............Myoglobin ..................Aspartate transcarbamylase,

R cha in*. ................Hemoglobin*. ..............&p coa t protein ..............Lysozyme ..................R17 coa t protein .............Ribonuclease ................Cytochrome c ..............Chymotrypsin*, 2 chains .....

*

Mel w t ofpolypeptide chain Reference. -220,000 9, 10, 11130,000 12100,000 13

94,000 12, 1468,000 15F3 ) 000 1G60,000 17, 1857,000 1953,000 12, 20, 2153 ) 000 Q50,000 2249,000 2343,000 2441,000 24 2jb41,000 21; 2640,000 24, 27, 2840,000 2937,000 3037,000 31 c3G, 000 32, 3336,000 13, 3436,000 2435,000 3534,00034, GO02!1,00027,60023,50025,70023,30023,00015,400 *17,20017,00015,50015,00014,30013,75013,70011,700

11,000 and 13,000

2, 336373822 c

ee. f

15 e

2, 3c400 c

e8ec

0 K. Weber, unpub lished results.b JORNVALL, H. AND HARRIS, J. I., Abstracts of the Federation ofEuropeaTL Bigchem ical Scienc es, Praha, 1968, abstract 759.c BUTLER, P. J. G. AND HARRIS, J. I., Abstracts of the Federationof European Bioche mical Scienc es, Praha, 1968, abstract 741.

d After performic acid oxidation.8 Calculated from the amino acid seque nces given in Dayhoff

and Eck (39).f Corrected according to the X-ray structure (J. Drenth, per-sonal communication).

0 W. Konigsberg, personal comm unication.

bygu

est,onMay7,2012

www.jbc.org

Downloadedfrom


4/7

IO I I I987 -1p: 6 c

I I0.2 0.4 0.6 0.8 1.0MobilityFI G. 2. 1)elerminatiou of the molecular weight of the poly-peptidc elmin of u-amino acid oxidase from a set o f 12 individualstnntl:t rcl gels. The six marker proteins rlsed were glrllamic aciddehydrogcnase, flmmrase, aldolase, glyceraldehydc phosphated&J-tlrogcnase, tryps ill, and m\-oglohin. All proteins were run ondllplic:~tc gels except glu~:~m~c dehydrogenase and myoglobin.The C~~WXUndicates the mobili(y of D-amino acid oxidase fromt\v o dillcrellt gels (0.398; 0.102). The molecular weights of themarker proteins are taken from Table I. The estrapolated valaefor I)-:mlilro acid oxidase is 37,000.

weipjit of namiiio ncid osidasc. A@1 the clectrophorcticmobili!ics for marker polypcl~~itlc chains are plotted xpaiust thelog of their niolccular w$hts. From the elcctrophorctic Inobilityof w:rlliiuo acid osidnse, a nlolccular weight of 37,000 ix found.

Ihc rclxoducibility of the I;>-atcm can be illustlatcd bh- thefollo\vilig cq)eriment. ~\sJKLrt:ltc tlalls:c:r~banl~-l:lsc Iron1 I$. coliKRS r1111n :I parallel I~:III~CI on 12 individual gels. Two pro&inbands \VCW: obtained which corrcsl)ond to the two differentpolylwl)tidc chains of this cnzync (2). The clcctrol)horcticmobilitics I\-crc 0.44 to 0.16 for the C Band and 0.73 to 0.76 forthe It 1i:md. The arcwgc mobilitics calculated from these 12gels \v(l rc 0.435 + 3c, :rlld 0.71~5 i 3L;$, rtsl )ccti \-cly. Fromthese rr~ults the rel)rotlllci})ilit?- a1q)cars better than 5:;. Ilo\\--PT-cr, 111~absolute ~~lucs of the rnobilit~~ of the ,samc pal)-l)cl)tidechain lu11 on selxuxte occxsions sometimes sholvs :L dcri:rtion of5 t,o 10;. This slight de\-iation scc~ns to occur with IIWV b:ltchesof l)ol!-ac.r!-larriiclc. Howcvcr, in ~11 GAYS, lhe plot of the mo-bilitiw for :L given set of stund:rrds gives thr sanic value for themolccul:~r weight of a lwticular lxotein.

The itlcntity of a particular lxotcin band has brcn rcl~atedlgestablished by the elution l~roccdure given in the l)rcriolw sec-tioll, lolloxcd by amino wid analysis. Greater than 75y0recovcry may be obtained by this method. The rncthotl :rlloc~-seasy sclwation and recovery 01 small quantities of lxotcins thatare suft icicnlly different in molwular x-eight.The lxoc*edure may bc :rd:~ptcd to study different molecularweight ranges. This GH~ be sccn iu Fig. 3. Six different polg-l)cl) t idc clGns were run wing 107; acrylamide solution, butchanging the amount of IIlcth?-lcnebisacr~l~lrllidc. ,L drasticchange in the mobilitics occurs. For example, the mobility of

IO987

glyceraldehyde phosphatedehydrogenase

02 0.4 06 08 IOMobility

FIG. 3. Ie has :L wriousde\-iation from the niolccular wciglits gi\.en ii1 lal)lc I beenshowll. The maximum deviation from the prctlic+xl \-aluw inthis ra~~gc i,* Iws thau 105, an d [or nlost, lxoteius the agreementis bcttcr . This accuracy is crlw;tcd bccausc, as sho\vl l ill Fig. t ,l)olyl)cl)tidc chains differing by oldy 105~1 in wcigllt wn beseparated on the same gel.Fig. 5 almwh the results for 10 diff crpnt l)olylwl)titlc chains inthe higllc~ nlolrculx weiglil wlrgc from 50,000 to 200,000. Gelswith h:l!f the ~~orn~~l an~oullt of c-rash-linker \YPI C 1~x1 ill thc,+ecspwitrwlit-. 111spite of the hylwbolic curve obtaiiwtl (xc alsoSh:rl)iro el rri. (1) and Fig. 3), the rnobilitics loilo~\- the, kno\~nn~olc~ul:tr \vcights . ALlthougll ~cwcr standards arc avuilablcfor this ixilgc, an accuracy of l IOL, still seems possible.

Our rcwltx shorv that, mwuingful molecular weights c:ln beobtained by 81)s gel elcctrol)horcsis for the three rnusclc pro-teins trolmtnyosin, paraniyosin, and inyosin. TfTe wvcrc interestedin these 1)roteins because they arc known to have a high helicalcolltcllt. Tropomyosin yields a molecular wight of 36,000 ingood :qgwment with the ~alucs given in the literature (13, 3-1).Using vccy low protciii concentrations we found that the 1)rolciriactually did not give a single band, but a doublet thal LV;LS nrdl~separated. At the present, it is unclear whether this is an artifactwith the lxcparation we hnvc available or mhcthcr the poly-peptide chains of tropomyosiii arc not identical. Iaramyosinyielded the expected pol~pcl~tide chain of molecular weight100,000 (13). For myosin KC obtained a polypeptitlc chain

bygu

est,onMay7,2012

www.jbc.org

Downloadedfrom


5/7

Molecular Weights by Gel Rlectrophoresis Vol. 244, No. 16

0.2 0.4 0.6 0.8 1.0MobilityFI G. 4. Comparison of the molecular weights of 37 different

polypeptide chain s in the molecular weight range from 11,000 to70,OOOwith their elcctrophoretic mo bilities on gels with the normalamount of cross-linker. The references to the moleclllar weightsare given in Table I.

with a molecular weight close to 200,000, in agreement with thereported values for this protein (9, 10). Howcvcr, we also de-tected three minor components of lower molecular weight,s(16;OOO to 23,000). At this time, we dont know if these com-ponents are inlpurities in the preparation KC had available or ifthey are inherent components of myosin. The latter assumptionis supported by recent results obtained in different laboratories.3In a few rases we studied the proteins after performic acidoxidation or carbox~meth~l:~tiol~. Th e electrophoretic mobil-ities of papain and ribonuclease after performic acid oxidationwere identical with the ones obtained before. Ko differenceswere seen after carbosymebhylation for lysozymc, aspartatetranscarballl3-lase, tropomyosin, and paramyosin. This indi-cates that the P-mercaptoethanol prevents the formation ofinterchain cyst ine bonds. This assumption is supported by thefac t that we did not find dimers of polypcptide chains. Thepossibility that some rare protein will not be delnatured by theSDS treatment in the presence of P-mercaptocthanol suggeststhat a control should be performed using an alkylated or oxidizedderivative.

3 S. Lowey, personal comm unication.

myosin

/.?-goloctosidase

phosphorylase aserum albuminL-amino acid oxidase

glutamic dehydrogenase

0.2 0.4 0.6 0.8 1.0MobilityFI G. 5. Comparison of the molecular weights of different

polypeptide chain s (see Table I) in the molec\llar weight rangefrom 40,000 to 200,000 with their electrophoretic mob ilities on gelswith half the amormt of cross-linker.

DISCUSSION

Separation of native proleins on polyacrylamidc gels wasshown by Ornstein (7) and T>avis (8) to be dependent not onlyon the charge but very strongly on t,he size of the molecules.The binding of dodecyl sulfate ions to proteins has been shownfor several protein molecules (see Tanford (41) for :I recent re-view), and was assumed to be the basis o f the separation of thedenatured proteins upon SDS electrophoresis on l)olyacrylamide(1). I f so, one must assume that the individual charge patternof each protein is tota lly changed by the binding of SDS anioix,rendering all molec ules negatively charged. At pre;ent it isdifficult to see why proteins that d iffer widely in amino acid com-position and isoelectric points should all follow the general pat-tern. It is possible that the sieving ef fect , which is an cxpo-nential function, overcomes the charge ef fect which may be ofminor importance. Because of these theoretical diff icult ies wewill d iscuss the results only from a practical point of view.

Two questions were raised during this study . How reproduc-ible are the results obtained by SDS gel electrophoresis and howreliable are the molecular weights obtained? The results pre-sented above quite clearly show that a high degree of reproduci-bility in the determination of the electrophorctic mobility can beobtained whether the proteins are run on dif ferent gels or on thesame gel.We have shown that close to 40 different prot,eins have clec-trophoretic mobilitics which are independent of the isoelectricpoint and the amino acid composition and seem governed solelyby the molecular weights of their polypeptide chains. Since themajor&y of the polypeptidc chains used are either characterizedby molecular weights known from amino acid sequence analysis,

bygu

est,onMay7,2012

www.jbc.org

Downloadedfrom


6/7

Issue of August 25, 1969 K. Weber and M. Osborn 4411or X-ray crystallography, or by very careful studies in guanidine-HCl with similar results obtained in diffe rent laboratories, wefeel confident that this study is a very stringent test for thetechnique of Shapiro et al. (1). All the proteins studied yieldedmolecular weights within the experimental error of the knownvalues. We have not studied proteins containing large amountsof carbohydrate or lipid material. It is possible that such pro-teins, because of their large nonprotein part, will behave dif -ferently on gel electrophoresis. It is, however, noteworthy thatSDS gel electrophoresis yields not only excellent results fo r pro-teins, which are globular in the native state, but also for thehighly helical, rod-shaped molecules, myosin, paramyosin, andtropomyosin. For all three proteins we obtained polypeptidechains with molecular weights in agreement with the valuesfrom the literature (see Results).

With these results one is inclined to assume that the techniqueof Shapiro et al. (1) yields molecular weights with an accuracy ofbetter than +lO% for polypeptide chains with molecular weightsbetween 15,000 and 100,000. This range can be covered withnumerous commercially available proteins as standards. Thedif ficult y with higher molecular weights is probably only thefac t. that fewer markers are available for the range 90,000 to200,000. Polypeptide chain markers commercially availablefor this range are phosphorylase with a molecular weight of 94,000(12, 14) and thyroglobulin with a molecular weight of 160,000(42). The preparation of P-galactosidase from E. coli can beeasily accomplished (43) and yields a marker band with 135,000molecular weight (12). Myosin and paramyosin can usually beobtained from the numerous laboratories working on these pro-teins and yield additional markers with molecular weights of200,000 and 100,000. With enough markers an accuracy ofabout 10% in the determination of the molecular weight of anunknown protein fall ing in this molecular weight range may bepossible.

Four examples will be used to illustrate these points further .Phosphorylase a gave a single polypeptide chain of molecularweight 94,000 without any indication of heterogeneity. This isin agreement with the results of Ullmann et al. (12), using theapproach to equilibrium centrifugation in the presence of 6 Mguanidine-HCl. Seery, Fischer, and Teller (14) found a similarvalue by equilibrium centrifugation in the same solvent onlyafter accounting for some heterogeneity and preferential hydra-tion. The latter, however, is an unlikely phenomenon in view ofthe results obtained by Kirby Hade and Tanford (44), Reitheland Sakura (45) and Ullmann et al. (12).A further sample is liver alcohol dehydrogenase. The tech-nique of Shapiro et al. (I) yields a value of 40,000 in agreementwith results obtained by osmotic pressure measurements inguanidine-HCl (24), X-ray analys is (25), and preliminary se-quence analysis4, but contrary t,o prior sedimentation analysisstudies (46).The separat,ion of the polypeptide chains of fumarase, aldolase,and glyceraldehyde phosphate dehydrogenase on one gel is verystriking. The molecular weights o f these polypeptide chains aretoday well established. Aldolase was for some time assumed tohave a molecular weight of 50,000. This value was mainlybased on preferential hydration in guanidine-HCl (47). How-ever by using glyceraldehyde phosphate dehydrogenase with aknown amino acid sequence as one marker and fumarase, which

4 J~~RNVALL, H., AND HARRIS, J. I., Abstracts of the Federation ofEuropean Biochemical Sciences, Praha, 1968, abstract 759.

is well characterized, as the other, it can be concluded that themolecular weight of the aldolase polypeptide chain is 40,000.Meanwhile, this is the value accepted for careful measurementsby ultracentrifugation (27) and osmotic pressure (24) in guani-dine-HCl, as well as from chemical studies (28).

Conflicting values for the size of the polypeptide chain ofn-amino acid oxidase are found in the literature and have beensummarized recently by Henn and Ackers (30). They showed avalue of 35,000 to 40,000 by gel filtra tion. The SDS electro-phoresis yields 37,000. From all the evidence presented for theother proteins we think that the two values indicating 37,000are much better than the one of 50,000 reported by Fonda andAnderson (48). Their value is based mainly on fingerprintanalysis and a gel filtrat ion study, which is less extensive thanthe one by Henn and Ackers (30).

Since the method appears to yield values in agreement with thebest current estimat,es for all the proteins studied, it seems fairto compare SDS gel electrophoresis with other methods. Theexcellent resolving power of the gels over a wide range of mo-lecular weights has been illustrated. In this respect the methodis much superior to gel filtration patterns on Sephadex in a de-naturing solvent like guanidine-HCl (49). The good resolutionand the fac t that an estimate of the molecular weight can beobtained within a day, together with the small amount of pro-tein needed, makes the method strongly competit ive with otherscommonly employed. The theoretically ful ly developed meth-ods of osmotic pressure and sedimentation equilibrium in 6M guanidine-HCl are still superior. However, osmotic pressuremeasurements suf fer from the large amount of protein neededand the fac t that extremely accurate protein determination isnecessary . Both limitations do not apply for sedimentationequilibrium using interference optics. However this method isvery demanding experimentally if good results are to be obtained.But even in the optimal case the calculation depends on thevalue chosen for the partial specif ic volume V. An uncertaintyof 0.02 in this value introduces a deviation of up to lo%, evenif all the centrifuge measurements are performed well. Also,in some cases, in spite of the fact that there is good evidence forno major change in ti in guanidine-HCl (12, 44, 45), the assump-tion of preferential hydration is still quite often used (14, 47) toaccount for deviations.It is by no means intended to assume that the accuracy ofSDS gel electrophoresis will be comparable with the well de-veloped physicochemical methods. Also, certain theoreticalaspects of SDS gel electrophoresis are still not clearly under-stood. In spite of these limitations, however, the ease withwhich the method can be applied, together with the resultspresented above, encourage its wider use, especially in case ofconflicting data obtained by other methods.

Acknowledgments-We thank Elizabeth Bloomquist for excel-lent and skillful assistance with many of these experiments.Discussions with Dr. R. Burgess, Dr. G. Guidotti, and AnnBurgess were most helpful.

REFERENCES1. SHAPIRO, A. L., VINUELA, E., AND MAIZEL, J. V., JR., Bio-

them. Biophys. Res. Commun., 28, 815 (1967).2. WEBER, K., Nature, 218, 1116 (1968).3. WILEY, D. C., AND LIPSCOMB, W. N., Nature, 218, 1119 (1968).4. WEBER, K., Biochemistry, 6, 3144 (1967).5. HIRS, C. H. W., J. Biol. Chem., 219, 611 (1956).

bygu

est,onMay7,2012

www.jbc.org

Downloadedfrom


7/7

Vol. 244, 50. 16,6.7.8.9.

10.11.12.13.14.15.10.li.18.19.20.21.22.

SEL.\, M., WHITE, F. H., .\NL) ASI'ISSEN, c. B., BiOChim.Bioph,+s. .I&, 31, 417 (1959).

OHSSTEIS, I,., ilnn. AT. Y. Acad. Sci., 121, 321 (19G-L).I).\vIs. 13. J., Ann. I\y. Y. A cad. Sri., 121, 404 (19G4).Wo ons , I:DELSTEIN, S. ,J., BiochenAby, 5, 2GSl(19GG).

48. FOND~I, M. I,., .\NU ASIIEILSO N, B. X., J. Biol. Ch(,rn., 243,5G35 (19G8); 244, GGG (1969).

49. N.\TH.\Ns, N. I)., J.;IZol. niol., 13, 521 (1965).

bygu

est,onMay7,2012

www.jbc.org

Downloadedfrom

Documents

Weber K. y Osborn M. 1969. the Reliability of Molecular Weight Determinations by Dodecyl