Vol. 43, No. 1INFECTION AND IMMUNITY, Jan. 1984, p. 127-1320019-9567/84/010127-06$02.00/0Copyright © 1984, American Society for Microbiology

Molecular Analysis of Immunoglobulins M and G Immune Responseto Protein Antigens of Treponema pallidum in Human Syphilis

MATTHAUS MOSKOPHIDIS AND FERDINAND MULLER*Division of Immunology, Department of Medical Microbiology, Institute of Hygiene, D-2000 Hamburg 36, West Germany

Received 8 July 1983/Accepted 13 October 1983

Protein antigens of Treponema pallidum precipitated by immunoglobulin M (IgM) and IgG antibodies ofsera from patients with untreated primary and secondary syphilis as well as treated secondary syphilis werecharacterized on a molecular basis. T. pallidum was labeled internally with [35S]methionine and solubilizedin 0.1% sodium dodecyl sulfate-1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophore-sis on 12.5% gels followed by autoradiography revealed 32 distinct proteins with molecular weights between13,500 and 200,000. Twenty-three proteins of T. pallidum with molecular weights between 15,500 and115,000 were identified as antigens by double antibody radioimmunoprecipitation with IgM and IgGantibodies of sera from syphilitic patients. The molecular analysis of the IgM and IgG immune response toT. pallidum in human syphilis is in accord with earlier immunological observations. Finally, utilizingsyphilitic human sera, we characterized 15 protein antigens of T. palliduim that are common to Treponemaphagedenis by partial absorption of IgM and IgG antibodies with an ultrasonicate of T. phagedenis.

Molecular investigations on the characterization of sur-face antigens using radiolabeled Treponema pallidum ex-tracts have shown that at least 11 outer membrane proteinswith molecular weights (MW) between 98,000 and 20,000react with antibodies of infected rabbits (1). Protein antigenswith MW of 89,000, 29,500, and 25,000 have been implicatedas ligands which attach themselves to the surface of eucary-otic host cells (3, 6). Lukehart et al. (7), using sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting (11), have identified atleast eight antigens of T. pallidum which react with a serumpool from infected rabbits. By the same technique, Hanff etal. (4, 5) have studied the humoral immune response toprotein antigens of T. pallidum in human syphilis.Methods hitherto used for antigen analysis (crossed elec-

trophoresis and SDS-PAGE followed by Western blotting)have different sensitivities (5). Theoretical considerationsled to the idea that internal radiolabeling of treponemes by[35S]methionine and subsequent separation of protein anti-gens by SDS-PAGE followed by autoradiography shouldresult in highly sensitive and specific findings as far asprotein antigens are concerned.Using this technique, our investigations were attempted to

determine whether distinct protein antigens of T. palliduminduce the synthesis of specific immunoglobulin M (IgM)and IgG antibodies in immunologically defined stages ofuntreated or treated human syphilis. It was intended further-more to identify those protein antigens of T. pallidum whichare no longer precipitated when syphilitic sera are absorbedby an ultrasonicate of Treponema phagedenis, i.e., proteinantigens which are common to both pathogenic (T. palliduim)and nonpathogenic (T. phagedenis) treponemes.

MATERIALS AND METHODSSource and radiolabeling of T. pallidum. T. pallidum Nich-

ols was inoculated intratesticulary in New Zealand Whiterabbits at 2 x 107 treponemes per testis. Treponemes wereharvested from tissue 8 to 11 days after infection by using anextracting medium containing 10 mM Na,HPO4, 4.7 mM

* Corresponding author.

KH2PO4, 20 mM thioglycollic acid, 1.1 mM cysteine, 2.5mM glutathione, 1.4 mM sodium pyruvate, 8.5 mMNaHCO3, 1.4 mM MgCl2, 103 mM NaCl, 20 mM glucose,and 2.5% (wt/vol) bovine serum albumin. The pH wasadjusted to 7.3 with NaOH. This medium was provided asatisfactory environment for radiolabeling of T. pallidium.Gross cell contaminants were removed from treponemes bycentrifugation at 500 x g for 10 min. The supernatantscontained approximately 108 treponemes per ml.Treponemes were incubated for 22 h at 35°C under an air

atmosphere in sterile polypropylene test tubes that con-tained 10 ml of medium and 150 ,uCi of [35S]methionine(specific activity, 1,200 Ci/mmol) or 150 puCi of [U-14C]pro-tein hydrolysate (specific activity, 56 mCi/milliatom of car-bon) (Amersham, Buckinghamshire, United Kingdom). Themotility of T. pallidum, examined by darkfield microscopyafter incubation for radiolabeling, was greater than 90%. Theorganisms were then pelleted by centrifugation at 15,000 x gfor 1 h, washed once in phosphate-buffered saline (PBS) (137mM NaCl, 2.7 mM KCI, 4.6 mM Na,HPO4, 1.5 mM KH-P04[pH 7.4]), pelleted, and stored at -70°C.Treponemes used for the demonstration of protein anti-

gens in gel electrophoresis by staining with Coomassiebrilliant blue were extracted with PBS (pH 7.4), followed bytwo differential centrifugations at 800 x g for 10 min.Supernatants containing 5 x 108 treponemes per ml weresubjected to Percoll (Pharmacia Fine Chemicals, Uppsala,Sweden) density gradient centrifugation at 30,000 x g for 30min at 4°C to remove host tissue contaminants. The bottomband was harvested and examined for purity by darkfieldmicroscopy. Treponemes were separated from Percoll bycentrifugation at 100,000 x g for 1 h at 4°C, washed threetimes in PBS (pH 7.4), pelleted by centrifugation at 15,000 xg for 30 min at 4°C, and stored at -70°C (7).

Sera. Sera were taken from patients with primary, second-ary, and treated secondary syphilis. A diagnosis of primarysyphilis was based on the presence of darkfield-positivegenital lesions (chancre) and reactive treponemal tests (T.pallidum hemagglutination assay and fluorescent treponemalantibody absorption [FTA-ABS] test). All patients withsecondary syphilis had a characteristic rash and/or palmarand plantar lesions, lymphadenitis, and reactive treponemal

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128 MOSKOPHIDIS AND MULLER

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FG1.Demonstration of IgM and IgG antibodies to T. pallidumin fractions after gel filtration of representative serum from patientswith primary syphilis. O, IgM-FTA-ABS; U, IgG-FTA-ABS; 0,concentration of total IgM; 0, concentration of total IgG.

and lipoidal tests. Patients with treated secondary syphilishad had secondary syphilis and were adequately treated withpenicillin 2 to 3 years before the investigation.

Sera from each of five patients in the above-mentionedstages of syphilis were examined separately for IgM and IgGantibodies. For molecular investigations the five sera of thethree groups of patients were pooled.

Separation and quantitation of IgM and IgG in sera.Separation of IgM from IgG antibodies in sera was carriedout by Ultrogel AcA 34 filtration (8). Briefly, 0.7 ml of activeserum was filtered through a gel column (1.5 by 14 cm) usingPBS (pH 7.3). Fractions of 1.3 ml were collected, and theabsorbance at 280 nm was measured. Quantitation of IgMand IgG in fractions was carried out by single radial immuno-diffusion using Partigen plates (Behringwerke, Marburg,West Germany).IgM- and IgG-FTA-ABS test. The technique for demon-

stration of IgM and IgG antibodies to T. pallidum after gelfiltration by indirect immunofluorescence has been de-scribed previously (8). The Nichols strain of T. pallidum wasused as antigen. For the detection of IgM or IgG antibodies,fluorescein isothiocyanate-labeled anti-human IgM (p.-chainspecific) or anti-human IgG (^y-chain specific) (Daco Immu-nochemicals, Copenhagen, Denmark) was used in a workingdilution of 1/50 for IgM or 1/200 for IgG.

Radioimmunoprecipitation assay. 3)5- or '4C-labeled T.pallidum was solubilized by the method of Baseman andHayes (3). Frozen pellets containing 10 radiolabeled trepo-nemes were suspended in 100 p.1l of TNE (50 mM Tris-hydrochloride, 150 mM NaCl, 5 mM EDTA [pH 7.4]) buffercontaining 2 mM phenylmethylsulfonyl fluoride. Ten micro-liters of 1%t SDS in TNE buffer was added, and the suspen-sion was gently homogenized until the treponemes weresolubilized. Then 100 p.1 of 10 mg of ovalbumin per ml inTNE buffer and 90 p.1 of 1% SDS in TNE buffer were added.Finally, 100 p.1 of 10% Triton X-100 was introduced, and themixture was incubated at 37°C for 30 mmn. The preparationwas diluted to a final volume of 1 ml in TNE buffer. Thesample was centrifuged at 100,000 x g for 1 h using aBeckman T 50 rotor to remove insoluble material. Thesupernatant was then used for the radioimmunoprecipitationassay.A double antibody radioimmune precipitation assay was

carried out as follows. Glass test tubes were coated by the

addition of 50 p. of 10% bovine serum albumin and 50 ,ul of5% Triton X-100; then 100 ,u1 of SDS-Triton X-100-soluble35S-labeled T. pallidum supernatants with approximately200,000 cpm and 20 ,u1 of human syphilitic or normal humanserum diluted with 80 ,u1 TNE buffer was introduced. Mix-tures were incubated for 18 h at 4°C, after which a volume ofrabbit antibodies to human IgM or IgG with p.- or -y-chainspecifity (Miles Laboratories, Elkhart, Ind.) sufficient toprecipitate more than 95% of the input of IgM or IgG wasadded. The second reaction was incubated at 37°C for 30 minand then at 4°C for 2 h. The precipitates formed were washedonce with TNE buffer containing 0.5% Triton X-100 and thentwice more with TNE buffer. The final pellet was dried anddissolved in 50 p. of solubilizing buffer (62.5 mM Tris-hydrochloride [pH 6.8], 5% ,B-mercaptoethanol, 2% SDS,10% glycerol, 0.02% bromphenol blue), followed by boilingfor 3 min. Cross-reacting antibodies of syphilitic sera to T.phagedenis were absorbed with an ultrasonicate of T. phage-denis (Hoffmann-La Roche, Basel, Switzerland). Lyophi-lized ultrasonicate (25 mg) was added to 100 p.1 of syphiliticserum. The suspension was incubated at 37°C for 30 min,and the ultrasonicate was removed by centrifugation at150,000 x g for 1 h.Gel electrophoresis and autoradiography. Discontinuous

SDS-slab gel electrophoresis was performed with stackingand separating gels of 3 and 12.5% acrylamide, respectively.Radioactively labeled samples that contained bromphenolblue as tracking dye were added in 50-,ul amounts to individ-ual slots in the stacking gel of the acrylamide slab. A mixtureof 0.1% SDS and 1% Triton X-100 containing samples ofsoluble 35S- or 14C-labeled T. pallidum was diluted 1/2 withsolubilizing buffer for gel electrophoresis.

Electrophoresis was initially performed with a constantcurrent of 24 mA, which was reduced to 14 mA when thetracking dye entered the separating gel. Slab gels were fixedand either stained with Coomassie blue or processed forautoradiography with Kodak X-Omat AR X-ray film at-700C.The protein mixture (Pharmacia, Uppsala, Sweden) or the

14C-methylated protein mixture (Amersham) was used as aMW marker. Approximate MW was determined by themethod of Weber and Osborn (12).

RESULTSIgM and IgG antibodies to T. pallidum in fractionated sera.

After gel filtration of sera, the fractions were investigated

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IMMUNE RESPONSE TO T. PALLIDUM 129

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FIG. 3. Demonstration of IgM and IgG antibodies to T. pallidurmin fractions after gel filtration of representative serum from patientswith treated secondary syphilis. For symbols. see the legend to Fig.1.

quantitatively by the IgM- and IgG-FTA-ABS. In fractionsof sera from five patients with primary syphilis, treponemalIgM antibodies with high titers and IgG antibodies in smallamounts were detected (Fig. 1). In fractions of sera fromsecondary syphilis, IgM and IgG antibodies were present inalmost equal amounts in all patients (Fig. 2). Only IgGtreponemal antibodies were demonstrated in fractions of fivesera from patients with treated secondary syphilis (Fig. 3).

Molecular characterization of T. pallidum proteins. Thetotal protein profile of T. pallidiitn is shown in Fig. 4. SDS-PAGE of T. pallidlin on a 12.5% gel after staining withCoomassie blue (Fig. 4A) revealed at least 35 distinctproteins; 35S- (Fig. 4C) or "4C-labeled (Fig. 4D) T. pallidiinrevealed 32 proteins between 15,500 and 200,000 with mostbands lying between MW 15,500 and 115,000. No significantdifference in the protein profiles between 35S- and '4C-

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itated by IgM antibodies followed by autoradiography. Primary (aand b), secondary (c and d), and treated secondary syphilis (e and f)before and after absorption of serum pools with T. phalgedenisultrasonicate. g, Marker proteins, MW x 103; h, total protein profileof 35S-labeled T. pallidum.

labeled T. pallidium could be observed. Both the Coomassieblue-stained and the 35S- and '4C-labeled gel profiles of T.pallidium showed identical patterns.

Protein antigens of T. pallidum precipitated by IgM antibod-ies. Reactions of IgM in pooled serum from patients withprimary syphilis to 35S-labeled T. pallidirn proteins areshown in Fig. Sa. The serum pool contained IgM antibodies,mainly to proteins with MW of 30,500, 33,000, 35,000, and37,000 (Table 1).The reaction of IgM in pooled serum from patients with

secondary syphilis to 35S-labeled proteins of T. pailliduin isshown in Fig. Sc. The serum pool contained IgM antibodiesto a wide spectrum of proteins with MW between 15,500 and115,000, with strong reactions to proteins with MW of15,500, 18,000, 30,500, 33,000, 35,000, 37,000, 38,500,40,000, 43,500, 46,000, and 49,500 (Table 1).

In the serum pool from patients with treated secondarysyphilis, a weak reaction with one protein antigen of MW33,000 was observed (Fig. Se). The same reaction wasdetected when pooled normal human serum was used (notshown).

Protein antigens of T. pallidum precipitated by IgG antibod-ies. Reactions of IgG in pooled serum from patients withprimary syphilis to 35S-labeled T. palliduiin proteins areshown in Fig. 6a. A weak reaction to proteins with MW of30,500, 33,000, and 37,000 was detected (Table 2).

Reactions of IgG in pooled serum from patients withsecondary syphilis to 35S-labeled proteins of T. palliduiini areshown in Fig. 6c. The serum pool contained antibodies toproteins of T. pallidiumii with the same wide spectrum andstrong reactions as with IgM between MW 15,500 and115,000 (Table 2).IgG of pooled serum from patients with treated secondary

syphilis showed weaker reactions to proteins of T. palliduinothan IgG from patients with untreated secondary syphilis. A

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130 MOSKOPHIDIS AND MULLER

TABLE 1. Total protein profile of T. pallidum and treponemal protein antigens precipitated by IgM antibodies in pooled serum frompatients with syphilis

Total protein Precipitated treponemal protein antigens"profile of

MW (x10) [35S]methionine- Primary Secondary Treated Treatedlabeled T. Primary absorbed Secondary absorbed secondary secondarypalliduin absorbed

200 +140 +115 +++ + +94 ++ + +79 +++ ++ +73 + + ++

69 + + ++ +63 +++ ++ ++59 +++ ++ ++53 ++49.5 +++ +++ ++46 + + + ++

43.5 +++ ++± ++40 + +++++ ++

38.5 +++ + + +++37 +++ +++ ++ ++

35 +++ +++ ++ +±+ ++

33 +++ +++ ++ +++ ++ + +

30.5 +++ ++ + +++ ++29 +++ ++ ++28 +++ ++26 +++ ++ +24.5 +24 +22.5 +++ ++21.5 +19.5 +18 ++ ++ ++16.5 +++ ++ ++15.5 +++ +++14.5 +13.5 +

" Reactivity of 1gM with individual T. pallidiin protein antigens: + ++. strong; + intermediate; +wWeak.

wide spectrum of proteins of T. palliditin between MW15,500 and 115,000 was detected, with strong reactions toproteins with MW of 30,500, 33,000, 35,000, and 37,000, ascan be seen in Fig. 6e and Table 2.Two weak precipitated bands with MW of 30,000 and

33,000 were detected when pooled normal human serum wasused (not shown).

Cross-reacting IgM and IgG antibodies of syphilitic sera toT. phagedenis. Cross-reacting IgM and IgG antibodies to T.phagedenis of pooled sera from patients with primary,secondary, and treated secondary syphilis were absorbedwith a lyophilized ultrasonicate of T. phagedenis. In Fig. 5the protein patterns of 35S-labeled T. pallidiin antigensprecipitated with IgM antibodies of an absorbed serum poolfrom patients with primary (Fig. Sb), secondary (Fig. Sd),and treated secondary syphilis (Fig. Sf) are shown. T.phagedenis ultrasonicate completely or partly absorbed IgMantibodies to proteins of T. pallidium with MW of 15,500,22,500, 26,000, 28,000, 30,500, 33,000, 35,000, 37,000,38,500, 40,000, 43,500, 46,000, 49,500, 69,000, and 79,000(Table 1).

In Fig. 6 the protein patterns of 35S-labeled T. pallidiumantigens, precipitated with IgG antibodies of pooled serafrom patients with primary (Fig. 6b), secondary (Fig. 6d),and treated secondary syphilis (Fig. 6f) after absorption ofsera with an ultrasonicate of T. phagedenis, are shown. Itcan be seen that IgG antibodies to proteins of T. pallidiiin

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itated by IgG antibodies followed by autoradiography. Primary (aand b), secondary (c and d), and treated secondary syphilis (e and f)before and after absorption of pooled sera with T. phagedenisultrasonicate. g, Marker proteins, MW x 103; h, total protein profileof 35S-labeled T. pallidum.

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TABLE 2. Protein antigens of T. pallidtrn precipitated by lgG antibodies in pooled serum from patients with syphilis

Precipitated treponemal protein antigens"

MW (x 1O') Primary Secondary Treated TreatedPrimary abobdSecondary abobdscnaysecondaryabsorbed

115 + +94 + +79 ++ + ++73 ++ ++69 ++ + ++63 ++ ++59 ++ ++49.5 +++ +±+ +46 +±+-+ +±+ +43.5 +++ ++40 +++ ++38.5 +++ ++ ++ +37 + + + + + + + + ++35 +++ ++ +++ ++33 + + +++ ++ +±++ ++30.5 + + +++ ++ +++ ++29 ++ ++ + +28 +±+ +26 ++ + +22.5 ++ +18 ±± ++16.5 ++ +-+15.5 +a+-+ +a+

" Reactivity of IgG with individual T. paillidium protein antigens: ++ strong, +a. intermediate; a. weak.

with the same MW as described for IgM antibodies werecompletely or partially absorbed (Table 2).

DISCUSSIONSpecific immunological assays for T. pallidium infections

(IgM-FTA-ABS and IgG-FTA-ABS) allow the demonstra-tion of IgM and IgG antibodies in fractions after gel filtrationof sera from patients with syphilis. Both IgM and IgGantibodies to T. pallidum were demonstrated in sera frompatients with primary and secondary syphilis. In primarysyphilis, IgM antibodies predominate over those of the IgGclass. In sera from patients with treated secondary syphilis,only IgG antibodies were found.IgM antibodies to T. pallidium can persist for several years

or even decades in the serum of patients with untreatedlatent syphilis. After antibiotic treatment, specific IgM anti-bodies disappear (2, 8, 9).

Using the highly sensitive internal labeling of T. pallidiumwith [35S]methionine, 32 proteins were differentiated bySDS-PAGE. All these proteins (except the very weak bands)were precipitated by IgM and IgG antibodies of sera frompatients with secondary syphilis and identified as antigens ofT. pallidium. Extending the investigations of Hanff et al. (4),who used the Western blotting technique, we identified fourmore protein antigens of T. pallidium (MW 37,000, 38,500,59,000, and 69,000) with strong activity.Our investigations lead to the conclusion that the humoral

immune response against T. pallidium starts with the synthe-sis of IgM antibodies to T. pallidium proteins with MW of30,500, 33,000, 35,000, and 37,000 (primary syphilis). In thenext stage of infection (secondary syphilis) IgM and IgGantibodies to all proteins of T. pallidlim detected by SDS-PAGE are synthesized. Antibodies to T. pallidiun proteinswith MW of 15,500, 18,000, 30,500, 33,000, 35,000, 37,000,38,500, 40,000, 43,500, 46,000, and 49,000 predominate. Insera from patients with treated secondary syphilis only IgG

antibodies were found, mainly to proteins of T. palliduinwith MW of 15,500, 30,500, 33,000, 35,000, and 37,000.

Utilizing sera from syphilitic patients and the Westernblotting technique, Hanff et al. (5) recently characterizedeight protein antigens with MW of 30,000, 33,000, 35,500,40,000, 45,000, 47,000, 73,000, and 82,000 that are commonto both T. pallidum and T. phagedenis biotype Reiter. Usinga crossed immunoelectrophoresis technique, Pedersen et al.(10) have identified six cross-reacting protein antigens. Fi-nally, Lukehart et al. (7) have defined five common antigensusing pooled rabbit anti-T. pallidum serum and the Westernblotting technique. Extending these observations, we foundsix more protein antigens of T. pallidum with MW of 15,500,22,500, 26,000, 28,000, 37,000, and 43,500 that are commonto T. phagedenis.For the induction of humoral and cellular immune re-

sponses and for the attachment to the surface of eucaryotichost cells, glycoproteins on the surface of virulent microor-ganisms play an important role. Further molecular investiga-tions should therefore be performed to permit the determina-tion of localization and the glycosylation status of identifiedprotein antigens of T. palliduin.

LITERATURE CITED

1. Alderete, J. F., and J. B. Baseman. 1980. Surface characteriza-tion of virulent Treponema pallidulm. Infect. Immun. 30:814-823.

2. Atwood, W. G., and J. L. Miller. 1966. Fluorescent treponemalantibodies in fractionated syphilitic sera. The immunoglobulinclass. Arch. Dermatol. 100:763-769.

3. Baseman, J. B., and E. C. Hayes. 1980. Molecular characteriza-tion of receptor binding proteins and immunogens of virulentTreponema pallidum. J. Exp. Med. 151:573-586.

4. Hanif, P. A., T. E. Fehninger, J. N. Miller, and M. A. Lovett.1982. Humoral immune response in human syphilis to polypep-tides of Treponema pallidum. J. Immunol. 129:1287-1291.

5. Hanff, P. A., J. N. Miller, and M. A. Lovett. 1983. Molecular

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characterization of common treponemal antigens. Infect. Im-mun. 40:825-828.

6. Hayes, N. S., K. E. Muse, A. M. Collier, and J. B. Baseman.1977. Parasitism by virulent Treponema pallidum of host cellsurfaces. Infect. Immun. 17:174-186.

7. Lukehart, S. A., S. A. Baker-Zander, and E. R. Gubish, Jr.1982. Identification of Treponema pallidum antigens: compari-son with a nonpathogenic treponeme. J. Immunol. 129:833-838.

8. Muller, F. 1982. The 19S (IgM)-FTA-ABS test in the serodiag-nosis of syphilis. Immun. Infekt. 10:23-34.

9. O'Neill, P., and C. S. Nicol. 1972. IgM class antitreponemalantibody in treated and untreated syphilis. Br. J. Vener. Dis.

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48:460-466.10. Pedersen, N. S., N. H. Axelsen, B. B. Jorgensen, and C. S.

Petersen. 1980. Antibodies in secondary syphilis against five offorty Reiter treponeme antigens. Scand. J. Immunol. 11:629-633.

11. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl. Acad. Sci.U.S.A. 76:4350-4354.

12. Weber, K., and M. Osborn. 1969. The reliability of molecularweight determinations by dodecyl sulfate polyacrylamide gelelectrophoresis. J. Biol. Chem. 244:4406-4412.

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