Measurement of Molecular Weights by Electrophoresis on SDS-Acrylamide Gel

Methods in Enzymology

Volumn 26, Issue C, 1972, Pages 3-27

  Weber, K ^a   Pringle, J R ^a   Osborn, M ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

AMINO ACID; DODECYL SULFATE SODIUM; ENZYME; PEPTIDE; PEPTIDE FRAGMENT; PROTEIN;

ANTIGEN ANTIBODY REACTION; ARTICLE; COST; METABOLISM; MOLECULAR WEIGHT; POLYACRYLAMIDE GEL ELECTROPHORESIS; TECHNIQUE;

AMINO ACIDS; ANTIGEN-ANTIBODY REACTIONS; COSTS AND COST ANALYSIS; ELECTROPHORESIS, POLYACRYLAMIDE GEL; ENZYMES; EVALUATION STUDIES; METHODS; MOLECULAR WEIGHT; PEPTIDE FRAGMENTS; PEPTIDES; PROTEINS; SODIUM DODECYL SULFATE;

EID: 0015437962     PISSN: 00766879     EISSN: 15577988     Source Type: Book Series    
DOI: 10.1016/S0076-6879(72)26003-7     Document Type: Article

References (49)

1. Shapiro, A.L., Viñuela, E, Maizel, J.V. Jr., Biochem. Biophys. Res. Commun., 28, 1967, 815.
2. Weber, K, Osborn, M, J. Biol. Chem., 244, 1969, 4406.
3. 17.
4. Wolf, B, Lausarot, P.M., Lesnaw, J.A., Reichmann, M.E., Biochim. Biophys. Acta, 200, 1970, 180.
5. Davies, G.E., Stark, G.R., Proc. Nat. Acad. Sci. U.S., 66, 1970, 651.
6. Pringle, J.R., Biochem. Biophys. Res. Commun., 39, 1970, 46.
7. Pringle, J.R., Ph.D. Thesis, 1970, Harvard University, Cambridge, Massachusetts.
8. Reynolds, J.A., Tanford, C, Proc. Nat. Acad. Sci. U.S., 66, 1970, 1002 We have used buffers of various ionic strengths, and with pH values in the range 4–9, without apparent difficulty. Ammonium sulfate at concentrations as high as 10% of saturation has had no ill effects. If appreciable concentrations of potassium, guanidinium, or other SDS-precipitating cations are present, dialysis against 0.01 M sodium phosphate, pH 7.0, is necessary. Buffers of high ionic strength can decrease SDS binding.
9. See C. H. W. Hirs, Vol. XI [19].
10. See A. M. Gold, Vol. XI [83]. PMSF can be obtained from Mann. It is much less poisonous than diisopropyl fluorophosphate, which can be used instead.
11. Vallee, B.L., Boyer, P.D., Lardy, H, Myrbäck, K, (eds.) The Enzymes, Vol. 3, 1960, Academic Press, New York, 259 1,10-Phenanthroline can be obtained from Sigma.
12. See F. R. N. Gurd, Vol. XI [62]; and J. F. Riordan and B. L. Vallee, Vol. XI [63].
13. See Maizel, J.V. Jr., Polyacrylamide Gel Electrophoresis of Viral Proteins. Maramorosch, K, Koprowski, H, (eds.) Methods in Virology, Vol. 5, 1971, Academic Press, New York.
14. Frazekas de St. Groth, S, Webster, R.G., Datyner, A, Biochim. Biophys. Acta, 71, 1963, 377.
15. Some workers [e.g., A. L. Shapiro, E. Viñuela, and J. V. Maizel, Jr., Biochem. Biophys. Res. Commun.28, 815 (1967)] have fixed the gels for 12–24 hours in 20% sulfosalicylic acid or in 10% trichloroacetic acid before staining. We omit this step without apparent problems. However, it may be advisable in the case of proteins that are slightly soluble in the staining solution. Such behavior might be expected for very small molecules.
16. Chrambach, A, Reisfeld, R.A., Wyckoff, M, Zaccari, J, Anal. Biochem., 20, 1967, 150 In this procedure the gel is immersed in 0.05% Coomassie Brilliant Blue in 10% trichloroacetic acid. The dye is relatively insoluble in this solution, and therefore partitions into protein-dye complexes. If enough protein is present in each band, the pattern will appear immediately after a short staining procedure, and no separate destaining step is necessary.
17. Ward, S, Anal. Biochem., 33, 1970, 259 The electrophoretic destaining method can, however, remove some small polypeptides [R. T. Swank and K. D. Munkres, Anal. Biochem.39, 462 (1971)]. Our transverse electrophoresis apparatus is modified from that described by Ward.
18. See W. R. Gray, Vol. XI [12].
19. Zacharius, R.M., Zell, T.E., Morrison, J.H., Woodlock, J.J., Anal. Biochem., 30, 1969, 148 On SDS gels this procedure can sometimes stain protein bands containing no carbohydrate. Care must be taken to eliminate SDS from the gel, including SDS bound to protein, and to keep the gel during staining at acid pH. A suitable procedure has been described [H. Glossman and D. N. Neville, Jr., J. Biol. Chem.246, 6339 (1971)]. A good control is to omit the periodate treatment—under such conditions carbohydrate will not stain.
20. Rice, R.H., Means, G.E., J. Biol. Chem., 246, 1971, 831.
21. Helleiner, C.W., Wunner, W.H., Anal. Biochem., 39, 1971, 333.
22. Fairbanks, G, Levinthal, C, Reeder, R.H., Biochem. Biophys. Res. Commun., 20, 1965, 393.
23. Greenwood, F.C., Hunter, W.M., Glover, J.S., Biochem. J., 89, 1963, 114.
24. Swank, R.T., Munkres, K.D., Anal. Biochem., 39, 1971, 462.
25. Laemmli, U.K., Nature, 227, 1970, 680.
26. J. V. Maizel and U. K. Laemmli, in preparation.
27. Pitt-Rivers, R, Impiombato, F.S.A., Biochem. J., 109, 1968, 825.
28. Reynolds, J.A., Tanford, C, Proc. Nat. Acad. Sci. U.S., 66, 1970, 1002.
29. Reynolds, J.A., Tanford, C, J. Biol. Chem., 245, 1970, 5161.
30. Fish, W.T., Reynolds, J.A., Tanford, C, J. Biol. Chem., 245, 1970, 5166.
31. Bretscher, M.S., Nature New Biology, 231, 1971, 229.
32. Arndt, D.J., Berg, P, J. Biol. Chem., 245, 1970, 665.
33. Tung, J.-S., Knight, C.A., Biochem. Biophys. Res. Commun., 42, 1971, 1117.
34. K. Weber, unpublished results.
35. Dunker, A.K., Rueckert, R.R., J. Biol. Chem., 244, 1969, 5074.
36. Strauss, E.G., Kaesberg, P, Virology, 42, 1970, 437 Another example of the influence of primary structure on mobility is seen with hemoglobin. On 12.5% acrylamide gels rabbit and duck hemoglobin give two bands corresponding to the separated α and β chains; under the same conditions mouse hemoglobin gives a single band with the mobility of the rabbit α chain. [M. Osborn and M. Mathews, unpublished results.].
37. Alternatively, the “split-gel” technique allows two sets of proteins to be run separately on the same gel [R. R. Traut, P. B. Moore, H. Delius, H. Noller, and A. Tissières, Proc. Nat. Acad. Sci. U.S.57, 1294 (1967)].
38. Gorovsky, M.A., Carlson, K, Rosenbaum, J.L., Anal. Biochem., 35, 1970, 359.
39. J. Kyte, personal communication, 1969.
40. Rosenbusch, J.P., Weber, K, J. Biol. Chem., 246, 1971, 1644.
41. Weber, K, Kuter, D.J., J. Biol. Chem., 246, 1971, 4504.
42. Weiner, A.M., Platt, T, Weber, K, J. Biol. Chem., 247, 1972, 3242.
43. K. Weber, T. Platt, and A. M. Weiner, unpublished results, 1971.
44. Horowitz, M.S., Scharff, M.D., Habel, K, Salzman, N.P., (eds.) Fundamental Techniques in Virology, 1969, Academic Press, New York, 253 ff.
45. Horowitz, M.S., Scharff, M.D., Habel, K, Salzman, N.P., (eds.) Fundamental Techniques in Virology, 1969, Academic Press, New York, 297 ff.
46. J. Rosenbusch and K. Weber, manuscript in preparation.
47. Schubert, D, Cohn, M, J. Mol. Biol., 53, 1970, 305.
48. Platt, T, Miller, J.H., Weber, K, Nature (London), 228, 1970, 1154.
49. Platt, T, Weber, K, Garen, D, Miller, J.H., Proc. Natl. Acad. Sci. U.S., 69, 1972, 897.