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A sensitive method for staining proteins transferred to nitrocellulose sheets
ANALYTICAL BIOCHEMISTRY
129, 486-489 (1983)
A Sensitive Method for Staining Proteins Transferred to Nitrocellulose Sheets ZBIGNIEW WOJTKOWIAK,l ROBERT C. BRIGGS, AND LUBOMIR S. HNILICA Z Department ofBiochemistry and the A. B. Hancock. Jr.. Memorial Laboratory ofthe Vanderbilt University Cancer Center, Vanderbilt University School ofMedicine. Nashville, Tennessee 37232
Received September 9, 1982 KEy WORDs: immunotransfers; nitrocellulose; electrophoresis; dinitrofluorobenzene; peroxidase-antiperoxidase.
A simple method for the visualization of polypeptides transferred from polyacrylamide gels to nitrocellulose sheets is described. The transferred proteins are reacted with 2,4 dinitrofluorobenzene (DNFB),3 incubated with antiserum to dinitrophenol (DNP), and visualized by using the peroxidase-antiperoxidase reagent method. The DNFB method is approximately 100 times more sensitive than the Coomassie brilliant blue or amido black staining. The transfer of electrophoretically resolved proteins to nitrocellulose sheets, followed by specific immunodetection of reactive antigens, was first described by Towbin et al. (I) and adapted for peroxidase-antiperoxidase staining (2) in our laboratory (3). Routinely, nitrocellulose sheets containing the transferred proteins are stained with amido black if the visualization of all proteins is desired. However, this staining procedure is relatively insensitive, and frequently antigenic proteins that are present in amounts beyond the detection of the amido black method can be detected immunochemically. A highly sensitive staining procedure which employs the formation of protein complexes I Present address: Department of Biochemistry, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland. 2 To whom all correspondence should be addressed. 3 Abbreviations used: DNFB. 2,4-dinitrofluorobenzene; DNP, 2,4-dinitrophenyl; SDS, sodium dodecyl sulfate.
0003-2697/83/040486·04$03.00/0 Copyright © 1983 by Academic Press. Inc. AU rights of reproduction in any form reserved.
with silver was developed by several investigators (4-6) and is, in its recent modification (7), available commercially through the BioRad Laboratories. Although the silver procedure is a method ofchoice for staining polyacrylamide gels, it is difficult to use with nitrocellulose transfers. We present here an alternative method of equal sensitivity based on the immunochemical detection of DNP-derivatized proteins. MATERIALS AND METHODS
Novikoff ascites hepatoma chromatin, fibrinogen (Kabi, Sweden), and bovine serum albumin (Sigma) were used in the transfer staining experiments. The Novikoff hepatoma chromatin was purified from isolated nuclei (8) by the method of Bonner et al. (9) as modified in our laboratory (10) and stored frozen in 2 mM Tris-HCl buffer, pH 7.5 (approx I mg/ml as DNA). Polyacrylamide gel electrophoresis. Samples containing 1 mg of chromatin DNA were incubated with 20 fJ-g of DNase I (Worthington) in 10 mM Tris-HCI, 1 mM MgClz , pH 7.4 at 3rc for 1 hr. The digested samples were mixed with 0.9 vol of a solution containing 4.44% sodium dodecyl sulfate (50S) (Bio-Rad), 22.2% glycerol, 25 ,ug pyronine Y, 0.139 M Tris-HCI, pH 6.8, and 0.1 vol of 2mercaptoethanol and were heated 2 min in a boiling-water bath and left to stand overnight 486
lMMUNOCHEMICAL STAINING OF PROTEINS
at room temperature. Samples not containing DNA were mixed and heated without DNase I digestion. All samples were electrophoresed as described by Laemmli (11), using 3% stacking and 7.5% running gel. Resolved proteins were visualized by staining with Coomassie brilliant blue (12) or with silver nitrate (7). Using parallel gels, the electrophoretically separated proteins were transferred to nitrocellulose sheets (Millipore HAWP304FO, 0.45 J,tM) as described by Towbin et al. (I). Staining ofnitrocellulose transfers. Control nitrocellulose sheets with immobilized proteins were stained with amido black (0.1% solution in 45% methanol and 10% acetic acid and destained in 2% acetic acid solution in 90% methanol) or with silver nitrate solution as described by Merril et al. (7). However, the high background prevented the evaluation of the latter. The dinitrobenzene staining was accomplished by incubating the sheets in 0.00001 to 0.1% DNFB in solution containing 50 mM NaCH0 3 in 50% dimethyl sulfoxide (Me2S0) at room temperature for 10 min . The sheets were washed several times with 50 mM NaHC0 3 in 50% M~SO to remove excess DNFB. Longer incubation periods made the removal of excess DNFB more difficult. The transfers were washed with to mM sodium phosphate-buffered 0.14 M NaCl, pH 7.2 (PBS), 5 times (each wash 5 min), rocked in 3% bovine serum albumin-IO% heat-inactivated calf serum solution in PBS at 40°C for 1 h, and incubated at 4°C overnight in 1:200 diluted rabbit antiserum to DNP (Miles). The diluent was 3% bovine serum albumin-IO% calf serum in PBS. The transfers were then washed 4 times (5 min each) with PBS and incubated with goat anti rabbit IgG (Serasource), diluted I:40 in 3% bovine serum albumin-IO% calf serum in PBS, for 30 min at room temperature, washed in PBS (4 times, 5 min), and incubated for 30 min at room temperature with the peroxidase-antiperoxidase complex (Miles) diluted I: 100 with 3% bovine serum albumin- 10% calf serum in PBS. Following PBS washes (3 times, 5 min),
487
the peroxidase activity was detected by exposing the transfers to 0.3 mg/ml of 3,3'diaminobenzidine and 0.005 % H 20 2 in 50 mM Tris-HCI buffer, pH 7.5. Analytical procedures. Protein concentration was determined according to Lowry et al. (13), and DNA was assayed by the procedure of Burton (14). RESULTS
The Novikoffhepatoma chromosomal proteins separated electrophoretically and stained directly with Coomassie brilliant blue are shown in Fig. IA. As can be seen, this method can detect with confidence proteins representing 12.5 J,tg of chromatin as DNA in each gel. The immunochemical technique permits the detection of lesser quantities, depending on the concentration of DNFB in the incubating solution (Fig. 1B), with the limit of2.5 ug of chromatin as DNA in 0.0 1% DNFB. DNFB concentrations of 0.1% and higher were difficult to remove by washing the nitrocellulose sheets, which resulted in unacceptably high backgrounds. Variations of the anti-DNP antibody dilution in ranges from 1:100 to I:400 did not affect the intensity of staining . Low antibody dilutions also resulted in high backgrounds. As can be seen in Figs. IBand C, the immunochemical staining of electrophoretically transferred proteins is as sensitive as the silver-stain procedure shown in Fig. IC for directly stained geL We were unable to overcome the exceedingly high background levels when attempting to use this staining procedure on nitrocellulose transfers. To our knowledge, there is no acceptable method or no commercial source of silver stain suitable for nitrocellulose sheets. Both methods could detect chromosomal proteins in samples containing only 2.5 J,tg of chromatin as DNA. The sensitivity of the immunochemical procedure was further confirmed by experiments using purified protein standards. Figure 2 compares the staining of electrophoretically separated ' and transferred fibrinogen. While the Coomassie brilliant blue (gels, Fig. 2A) or amido black (transfers, Fig. 2B) are
488
WOJTKOWIAK, BRIGGS. AND HNILICA
c
B
A a
c
b
d
200K11 6 K94 K68 K-
43K-
M I
2
3
4
2
2
2
3
3 4
3 4
FIG. I. Polyacrylamide gel electrophoresis of Novikoff hepatoma chromatin. Samples at the indicated concentrations (as /lg of DNA) were electrophoresed and either stained directly with Coomassie brilliant blue (A), silver stain (C), or, after electrophoretic transfer to nitrocellulose sheets, with antibod y to DNP followed by the peroxidase-antiperoxidase procedure (B). (a-d) The concentrations of DNFB in the incubation mixture (see Materials and Methods), which were 0.0000 1,0.0001 ,0.001, and 0.01%, respectively. 1-4 = Chromatin concentrations applied to the gels (as /lg of DNA ): I = 62.5 /lg; 2 = 12.5 /lg; 3 = 2.5 /lg; 4 = 0.5 /lg; M = Molecuar weight markers (as indicated in the left margin).
A
comparable in detecting I Jlg of protein, the DNFB staining can detect lOng of protein (transfers, Fig. 2C). Similar results were obtained for bovine serum albumin (Figs. 3AC). Again, the immunochemical staining was nearly 100 times more sensitive than the amido black procedure.
c
B
DISCUSSION
Application of immunochemical techniques to the detection of individual antigens separated electrophoretically in polyacrylamide gels and transferred to nitrocellulose sheets (I) brought about the need for protein staining procedures which would match in sensitivity the autoradiography ofIabeled protein A or peroxidase-antiperoxidase staining
M I
2 3
4
2
3
4
2 3
4
of reactive antigens. Although the procedure
FIG. 2. Pol yacrylamide gel electrophoresis of co m mer-
developed for the detection of protein complexes with silver is both sensitive and relatively simple, its application is somewhat limited for staining proteins transferred to nitrocellulose sheets. The amido black stain, on
cial fibrinogen. (A) Coomassie brilliant blue stained gels. (B) Amido black stained nitrocellulose transfers. (C) DNP antibody-peroxidase-antiperoxidase stained nitrocellulose transfers. Each lane contained: I = 10 }!g; 2 = 1 /lg; 3 = 100 ng; and 4 = 10 ng of protein . M = Molecular weight markers (as indicated in Fig. I).
IM MU NOCH EMI CAL STA IN ING OF PR OT EI NS
A
B
c
489
likely explanation of this phenomenon is that small proteins present fewer DNFB-binding sites. However, at sufficient concentrations, all DNP proteins are detectable, making this procedure acceptable for their qualitative detection. ACKNOWLEDGMENTS Suppo rted by Na tio na l Can cer Institute Grants CA· 18389 and CA-264 12.
REFERENCES
M
I
2 3
4
23 4
2 3 4
FIG. 3. Polyacrylamide gel electrophoresis of commercial bovine serum albumin. (A) Co omassie brilliant blue sta ined gel. (B) Amide black stai ned nitrocellulose transfers. (C) DNP antibody- peroxidase-antiperoxidase stai ned nitrocellulose tran sfers. Each lane contained: 1 = 10 ug; 2 = I Ilg: 3 = 100 ng; and 4 = 10 ng of protein . M = M olecular weight mar kers (as indicated in Fig. 1).
the other hand, while quite suitable for this purpose, is about 100 times less sensitive than the comm ercially available silver stain. Immunochemical procedures are exceptionally sensitive, paralleling or surpassing chemical staining methods. It should be possible, therefore, to detect prot eins which were first modified by immunoreactive reagent by specific antiserum to the latter. The described dinitrophenylation procedure fulfills this requirement; it derivatized all tested proteins, making them detectable by antibody to DNP. The sensitivity of this immunodetection procedure equals or surpasses that of the silver stain. As our experiments with Novikoff hepatoma chromatin indicate, at the lowest range of detection, low M, proteins (approx M, 60,000 and lower) did not stain well. The most
I. T owbin , H., Staehelin , T. , and Gordon , J. (1979) Proc. Nat. Acad . Sci. USA 76, 4350-4355 . 2 . St ernberger, L. A. ( 197 9) in Immun ochemistry (Stern berger, 1. A., ed.), pp . 104-1 69, Pren tice Hall, Engelwood Cliffs, N. J. 3. G lass, W. F., Briggs, R. C, and Hn ilica, L. S. ( 198 1) Science 21I, 70-72. 4. Switzer, R. C, M erril , C R., and Shifrin, S. (1979) Anal. Biochem. 98 , 23 1-237. 5. Oa kley, B. R., Kirsch, D. R., and Mo rris, N. R. ( 1980) Anal. Biochem. lOS, 36 1-363. 6. Wr ay, W. , BouIikas, T ., Wray , V. P., and Hancock, R. (1981) Anal . Biochem. 118, 197-203. 7. Merril, C R., Goldman , 0. , Sedman, S. A., and Ebert, M. H. ( 1981) Science 211, 1437- 1438. 8. Wilhelm , J. A., Ansevi n, A. T ., Johnson , A. W., and Hnilica, 1. S. (1972) Biochim. Biophys. Acta 272, 220- 230. 9. Bon ner , J., Chalkley, R. G ., Dahmus, M ., Fambrou gh, D., Fujimura , F., Huan g, R. C C , Hub-er man , J., Jensen , R., Marushige, K., Oh lenbusch , H., Olivera , B., and Widholm, J. (1968) in Meth ods in Enz ym ology (G rossman, L. , and Moldave, K.. eds.), Vol. 12, Part B, pp . 3-65 , Academic Press, New York. 10. Spelsberg, T. C , and Hnilica, L. S. (19 7 1) Biochirn. Biophys. Acta 228 , 202 -2 12. 11. L aemmli, U. K. ( 1970) Nature (London) 277, 680685 . 12. Fairba nks, T ., Steck, T. L., and Wallach , D. F. 1-1. (1971) Biochem istry 10, 2606-26 17. 13. Lowry, O. H ., Ro sebrough, N. J., Farr, A. L. , and Randall, R. J. ( 195 1) J. Bioi. Chem . 193, 265275. 14. Burt on , K . (1956) Biochem. J. 62, 315-323.
129, 486-489 (1983)
A Sensitive Method for Staining Proteins Transferred to Nitrocellulose Sheets ZBIGNIEW WOJTKOWIAK,l ROBERT C. BRIGGS, AND LUBOMIR S. HNILICA Z Department ofBiochemistry and the A. B. Hancock. Jr.. Memorial Laboratory ofthe Vanderbilt University Cancer Center, Vanderbilt University School ofMedicine. Nashville, Tennessee 37232
Received September 9, 1982 KEy WORDs: immunotransfers; nitrocellulose; electrophoresis; dinitrofluorobenzene; peroxidase-antiperoxidase.
A simple method for the visualization of polypeptides transferred from polyacrylamide gels to nitrocellulose sheets is described. The transferred proteins are reacted with 2,4 dinitrofluorobenzene (DNFB),3 incubated with antiserum to dinitrophenol (DNP), and visualized by using the peroxidase-antiperoxidase reagent method. The DNFB method is approximately 100 times more sensitive than the Coomassie brilliant blue or amido black staining. The transfer of electrophoretically resolved proteins to nitrocellulose sheets, followed by specific immunodetection of reactive antigens, was first described by Towbin et al. (I) and adapted for peroxidase-antiperoxidase staining (2) in our laboratory (3). Routinely, nitrocellulose sheets containing the transferred proteins are stained with amido black if the visualization of all proteins is desired. However, this staining procedure is relatively insensitive, and frequently antigenic proteins that are present in amounts beyond the detection of the amido black method can be detected immunochemically. A highly sensitive staining procedure which employs the formation of protein complexes I Present address: Department of Biochemistry, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland. 2 To whom all correspondence should be addressed. 3 Abbreviations used: DNFB. 2,4-dinitrofluorobenzene; DNP, 2,4-dinitrophenyl; SDS, sodium dodecyl sulfate.
0003-2697/83/040486·04$03.00/0 Copyright © 1983 by Academic Press. Inc. AU rights of reproduction in any form reserved.
with silver was developed by several investigators (4-6) and is, in its recent modification (7), available commercially through the BioRad Laboratories. Although the silver procedure is a method ofchoice for staining polyacrylamide gels, it is difficult to use with nitrocellulose transfers. We present here an alternative method of equal sensitivity based on the immunochemical detection of DNP-derivatized proteins. MATERIALS AND METHODS
Novikoff ascites hepatoma chromatin, fibrinogen (Kabi, Sweden), and bovine serum albumin (Sigma) were used in the transfer staining experiments. The Novikoff hepatoma chromatin was purified from isolated nuclei (8) by the method of Bonner et al. (9) as modified in our laboratory (10) and stored frozen in 2 mM Tris-HCl buffer, pH 7.5 (approx I mg/ml as DNA). Polyacrylamide gel electrophoresis. Samples containing 1 mg of chromatin DNA were incubated with 20 fJ-g of DNase I (Worthington) in 10 mM Tris-HCI, 1 mM MgClz , pH 7.4 at 3rc for 1 hr. The digested samples were mixed with 0.9 vol of a solution containing 4.44% sodium dodecyl sulfate (50S) (Bio-Rad), 22.2% glycerol, 25 ,ug pyronine Y, 0.139 M Tris-HCI, pH 6.8, and 0.1 vol of 2mercaptoethanol and were heated 2 min in a boiling-water bath and left to stand overnight 486
lMMUNOCHEMICAL STAINING OF PROTEINS
at room temperature. Samples not containing DNA were mixed and heated without DNase I digestion. All samples were electrophoresed as described by Laemmli (11), using 3% stacking and 7.5% running gel. Resolved proteins were visualized by staining with Coomassie brilliant blue (12) or with silver nitrate (7). Using parallel gels, the electrophoretically separated proteins were transferred to nitrocellulose sheets (Millipore HAWP304FO, 0.45 J,tM) as described by Towbin et al. (I). Staining ofnitrocellulose transfers. Control nitrocellulose sheets with immobilized proteins were stained with amido black (0.1% solution in 45% methanol and 10% acetic acid and destained in 2% acetic acid solution in 90% methanol) or with silver nitrate solution as described by Merril et al. (7). However, the high background prevented the evaluation of the latter. The dinitrobenzene staining was accomplished by incubating the sheets in 0.00001 to 0.1% DNFB in solution containing 50 mM NaCH0 3 in 50% dimethyl sulfoxide (Me2S0) at room temperature for 10 min . The sheets were washed several times with 50 mM NaHC0 3 in 50% M~SO to remove excess DNFB. Longer incubation periods made the removal of excess DNFB more difficult. The transfers were washed with to mM sodium phosphate-buffered 0.14 M NaCl, pH 7.2 (PBS), 5 times (each wash 5 min), rocked in 3% bovine serum albumin-IO% heat-inactivated calf serum solution in PBS at 40°C for 1 h, and incubated at 4°C overnight in 1:200 diluted rabbit antiserum to DNP (Miles). The diluent was 3% bovine serum albumin-IO% calf serum in PBS. The transfers were then washed 4 times (5 min each) with PBS and incubated with goat anti rabbit IgG (Serasource), diluted I:40 in 3% bovine serum albumin-IO% calf serum in PBS, for 30 min at room temperature, washed in PBS (4 times, 5 min), and incubated for 30 min at room temperature with the peroxidase-antiperoxidase complex (Miles) diluted I: 100 with 3% bovine serum albumin- 10% calf serum in PBS. Following PBS washes (3 times, 5 min),
487
the peroxidase activity was detected by exposing the transfers to 0.3 mg/ml of 3,3'diaminobenzidine and 0.005 % H 20 2 in 50 mM Tris-HCI buffer, pH 7.5. Analytical procedures. Protein concentration was determined according to Lowry et al. (13), and DNA was assayed by the procedure of Burton (14). RESULTS
The Novikoffhepatoma chromosomal proteins separated electrophoretically and stained directly with Coomassie brilliant blue are shown in Fig. IA. As can be seen, this method can detect with confidence proteins representing 12.5 J,tg of chromatin as DNA in each gel. The immunochemical technique permits the detection of lesser quantities, depending on the concentration of DNFB in the incubating solution (Fig. 1B), with the limit of2.5 ug of chromatin as DNA in 0.0 1% DNFB. DNFB concentrations of 0.1% and higher were difficult to remove by washing the nitrocellulose sheets, which resulted in unacceptably high backgrounds. Variations of the anti-DNP antibody dilution in ranges from 1:100 to I:400 did not affect the intensity of staining . Low antibody dilutions also resulted in high backgrounds. As can be seen in Figs. IBand C, the immunochemical staining of electrophoretically transferred proteins is as sensitive as the silver-stain procedure shown in Fig. IC for directly stained geL We were unable to overcome the exceedingly high background levels when attempting to use this staining procedure on nitrocellulose transfers. To our knowledge, there is no acceptable method or no commercial source of silver stain suitable for nitrocellulose sheets. Both methods could detect chromosomal proteins in samples containing only 2.5 J,tg of chromatin as DNA. The sensitivity of the immunochemical procedure was further confirmed by experiments using purified protein standards. Figure 2 compares the staining of electrophoretically separated ' and transferred fibrinogen. While the Coomassie brilliant blue (gels, Fig. 2A) or amido black (transfers, Fig. 2B) are
488
WOJTKOWIAK, BRIGGS. AND HNILICA
c
B
A a
c
b
d
200K11 6 K94 K68 K-
43K-
M I
2
3
4
2
2
2
3
3 4
3 4
FIG. I. Polyacrylamide gel electrophoresis of Novikoff hepatoma chromatin. Samples at the indicated concentrations (as /lg of DNA) were electrophoresed and either stained directly with Coomassie brilliant blue (A), silver stain (C), or, after electrophoretic transfer to nitrocellulose sheets, with antibod y to DNP followed by the peroxidase-antiperoxidase procedure (B). (a-d) The concentrations of DNFB in the incubation mixture (see Materials and Methods), which were 0.0000 1,0.0001 ,0.001, and 0.01%, respectively. 1-4 = Chromatin concentrations applied to the gels (as /lg of DNA ): I = 62.5 /lg; 2 = 12.5 /lg; 3 = 2.5 /lg; 4 = 0.5 /lg; M = Molecuar weight markers (as indicated in the left margin).
A
comparable in detecting I Jlg of protein, the DNFB staining can detect lOng of protein (transfers, Fig. 2C). Similar results were obtained for bovine serum albumin (Figs. 3AC). Again, the immunochemical staining was nearly 100 times more sensitive than the amido black procedure.
c
B
DISCUSSION
Application of immunochemical techniques to the detection of individual antigens separated electrophoretically in polyacrylamide gels and transferred to nitrocellulose sheets (I) brought about the need for protein staining procedures which would match in sensitivity the autoradiography ofIabeled protein A or peroxidase-antiperoxidase staining
M I
2 3
4
2
3
4
2 3
4
of reactive antigens. Although the procedure
FIG. 2. Pol yacrylamide gel electrophoresis of co m mer-
developed for the detection of protein complexes with silver is both sensitive and relatively simple, its application is somewhat limited for staining proteins transferred to nitrocellulose sheets. The amido black stain, on
cial fibrinogen. (A) Coomassie brilliant blue stained gels. (B) Amido black stained nitrocellulose transfers. (C) DNP antibody-peroxidase-antiperoxidase stained nitrocellulose transfers. Each lane contained: I = 10 }!g; 2 = 1 /lg; 3 = 100 ng; and 4 = 10 ng of protein . M = Molecular weight markers (as indicated in Fig. I).
IM MU NOCH EMI CAL STA IN ING OF PR OT EI NS
A
B
c
489
likely explanation of this phenomenon is that small proteins present fewer DNFB-binding sites. However, at sufficient concentrations, all DNP proteins are detectable, making this procedure acceptable for their qualitative detection. ACKNOWLEDGMENTS Suppo rted by Na tio na l Can cer Institute Grants CA· 18389 and CA-264 12.
REFERENCES
M
I
2 3
4
23 4
2 3 4
FIG. 3. Polyacrylamide gel electrophoresis of commercial bovine serum albumin. (A) Co omassie brilliant blue sta ined gel. (B) Amide black stai ned nitrocellulose transfers. (C) DNP antibody- peroxidase-antiperoxidase stai ned nitrocellulose tran sfers. Each lane contained: 1 = 10 ug; 2 = I Ilg: 3 = 100 ng; and 4 = 10 ng of protein . M = M olecular weight mar kers (as indicated in Fig. 1).
the other hand, while quite suitable for this purpose, is about 100 times less sensitive than the comm ercially available silver stain. Immunochemical procedures are exceptionally sensitive, paralleling or surpassing chemical staining methods. It should be possible, therefore, to detect prot eins which were first modified by immunoreactive reagent by specific antiserum to the latter. The described dinitrophenylation procedure fulfills this requirement; it derivatized all tested proteins, making them detectable by antibody to DNP. The sensitivity of this immunodetection procedure equals or surpasses that of the silver stain. As our experiments with Novikoff hepatoma chromatin indicate, at the lowest range of detection, low M, proteins (approx M, 60,000 and lower) did not stain well. The most
I. T owbin , H., Staehelin , T. , and Gordon , J. (1979) Proc. Nat. Acad . Sci. USA 76, 4350-4355 . 2 . St ernberger, L. A. ( 197 9) in Immun ochemistry (Stern berger, 1. A., ed.), pp . 104-1 69, Pren tice Hall, Engelwood Cliffs, N. J. 3. G lass, W. F., Briggs, R. C, and Hn ilica, L. S. ( 198 1) Science 21I, 70-72. 4. Switzer, R. C, M erril , C R., and Shifrin, S. (1979) Anal. Biochem. 98 , 23 1-237. 5. Oa kley, B. R., Kirsch, D. R., and Mo rris, N. R. ( 1980) Anal. Biochem. lOS, 36 1-363. 6. Wr ay, W. , BouIikas, T ., Wray , V. P., and Hancock, R. (1981) Anal . Biochem. 118, 197-203. 7. Merril, C R., Goldman , 0. , Sedman, S. A., and Ebert, M. H. ( 1981) Science 211, 1437- 1438. 8. Wilhelm , J. A., Ansevi n, A. T ., Johnson , A. W., and Hnilica, 1. S. (1972) Biochim. Biophys. Acta 272, 220- 230. 9. Bon ner , J., Chalkley, R. G ., Dahmus, M ., Fambrou gh, D., Fujimura , F., Huan g, R. C C , Hub-er man , J., Jensen , R., Marushige, K., Oh lenbusch , H., Olivera , B., and Widholm, J. (1968) in Meth ods in Enz ym ology (G rossman, L. , and Moldave, K.. eds.), Vol. 12, Part B, pp . 3-65 , Academic Press, New York. 10. Spelsberg, T. C , and Hnilica, L. S. (19 7 1) Biochirn. Biophys. Acta 228 , 202 -2 12. 11. L aemmli, U. K. ( 1970) Nature (London) 277, 680685 . 12. Fairba nks, T ., Steck, T. L., and Wallach , D. F. 1-1. (1971) Biochem istry 10, 2606-26 17. 13. Lowry, O. H ., Ro sebrough, N. J., Farr, A. L. , and Randall, R. J. ( 195 1) J. Bioi. Chem . 193, 265275. 14. Burt on , K . (1956) Biochem. J. 62, 315-323.