Activity Gels: Reformation of Functional Proteins in SDS-Polyacrylamide Gels

  • Robert P. Dottin
  • Bodduluri Haribabu
  • Clifford W. Schweinfest
  • Richard E. Manrow
Part of the Genetic Engineering book series (GEPM)


SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is an important technique for analyzing the complexity of polypeptides in crude mixtures and for determining the subunit structures of purified proteins (1). The two-dimensional gel system developed by O’Farrell, that combines isoelectric focusing in urea with SDS-PAGE, offers the additional advantage of greater resolution than SDS-PAGE alone (2,3). Even small amounts of proteins can be resolved as discrete spots, and microheterogeneity in related polypeptides can be easily detected. However, the power of these techniques is limited by the fact that the proteins remain denatured in the gel and only abundant proteins can be identified by their positions on the gels. One recent approach to the detection of nonabundant proteins in a crude mixture, Western blotting, is the use of antibodies to identify specific antigens after separation on SDS-gels and transfer to nitrocellulose paper (4). This technique, though powerful, has the limitation of requiring antibodies, which usually necessitates prior purification of a specific protein of interest. An alternative approach is to detect isoproteins in a gel by their biochemical activities. Our laboratory first developed these procedures for identification of enzymes in high-resolution denaturing gels and recently they have been employed for other enzymes.


Crude Extract Mixed Micelle Dodecyl Alcohol Adenylate Kinase Molecular Heterogeneity 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Laemmli, U.K. (1970) Nature 227, 680–685.CrossRefGoogle Scholar
  2. 2.
    O’Farrell, P.H. (1975) J. Biol. Chem. 250, 4007–4021.Google Scholar
  3. 3.
    O’Farrell, P.Z., Goodman, H.M. and O’Farrell, P.H. (1977) Cell 12, 1133–1142.CrossRefGoogle Scholar
  4. 4.
    Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Nat. Acad. Sci. U.S.A. 76, 4350–4354.CrossRefGoogle Scholar
  5. 5.
    Geahlen, R.L. and Harrison, M.L. (1986) Biochem. Biophys. Res. Commun. 134, 963–969.CrossRefGoogle Scholar
  6. 6.
    Manrow, R.E. and Dottin, R.P. (1982) Anal. Biochem. 120, 181–188.CrossRefGoogle Scholar
  7. 7.
    Karawya, E.M. and Wilson, S.H. (1982) J. Biol. Chem. 257, 13129–13134.Google Scholar
  8. 8.
    Blank, A., Silber, J.R., Thelen, M.P. and Dekker, C.A. (1983) Anal. Biochem. 135, 423–430.CrossRefGoogle Scholar
  9. 9.
    Thelen, M.R., Blank, A., McKeon, T.A. and Dekker, C.A. (1982) Fed. Proc. 41, 1203.Google Scholar
  10. 10.
    Karawya, E.M., Swack, J.A. and Wilson, S.H. (1983) Anal. Biochem. 135, 318–325.CrossRefGoogle Scholar
  11. 11.
    Betrazzoni, U., Scovassi, A.I., Mezzina, M., Sarasim, A., Franchi, E. and Izzo, R. (1986) Trends Genetics 2, 67–72.CrossRefGoogle Scholar
  12. 12.
    Lacks, S.A. and Springhorn, S.S. (1980) J. Biol. Chem. 255, 7467–7473.Google Scholar
  13. 13.
    Geahlen, R.L., Anostario, M., Jr., Low, P.S. and Harrison, M.L. (1986) Anal. Biochem. 153, 151–158.CrossRefGoogle Scholar
  14. 14.
    Tuszynski, G.P., Baker, S.R., Fuhrer, J.P., Buck, C.A. and Warren, L. (1978) J. Biol. Chem. 253, 6092–6099.Google Scholar
  15. 15.
    Blumenthal, J. and Landers, T.A. (1976) Biochemistry 15, 422–425.CrossRefGoogle Scholar
  16. 16.
    Dottin, R.P., Manrow, R.E., Fishel, B.R., Ackerman, S.L. and Culleton, J.L. (1979) Methods Enzymol. 68, 513–527.CrossRefGoogle Scholar
  17. 17.
    Manrow, R.E. and Dottin, R.P. (1980) Proc. Nat. Acad. Sci. U.S.A. 77, 730–734.CrossRefGoogle Scholar
  18. 18.
    Harris, H. and Hopkinson, D.A. (1977) Handbook of Enzyme Electrophoresis in Human Genetics, North Holland, Amsterdam.Google Scholar
  19. 19.
    Hebb, M.J. and Gabriel, O. (1984) Methods Enzymol. 104, 416–439.CrossRefGoogle Scholar
  20. 20.
    Coates, P.M., Mestriner, M.A. and Hopkinson, D.A. (1980) Ann. Human Genet. 39, 1.CrossRefGoogle Scholar
  21. 21.
    Heussen, C. and Dowdle, E.B. (1980) Anal. Biochem. 102, 196–202.CrossRefGoogle Scholar
  22. 22.
    Dimond, R.L., Farnsworth, P.A. and Loomis, W.F. (1976) Dev. Biol. 50, 169–181.CrossRefGoogle Scholar
  23. 23.
    Fishel, B.R., Manrow, R.E. and Dottin, R.P. (1982) Dev. Biol. 92, 175–187.CrossRefGoogle Scholar
  24. 24.
    Fishel, B.R., Ragheb, J.R., Rajkovic, A., Haribabu, B., Schweinfest, C.W. and Dottin, R.P. (1985) Dev. Biol. 110, 369–381.CrossRefGoogle Scholar
  25. 25.
    Manrow, R.E. (1981) Doctoral thesis, The Department of Biology, The Johns Hopkins University, Baltimore, MD.Google Scholar
  26. 26.
    Manrow, R.E., Fishel, B.R., Schweinfest, C.W. and Dottin, R.P. (1979) J. Cell. Biol. 83, 44a.Google Scholar
  27. 27.
    Spanos, A., Sedgwick, S.G., Tarranton, G.T., Hübscher, U. and Banks, G.R. (1981) Nucl. Acids Res. 9, 1825–1839.CrossRefGoogle Scholar
  28. 28.
    Spanos, A. and Hübscher, U. (1983) Methods Enzymol. 91, 263–277.CrossRefGoogle Scholar
  29. 29.
    Plevani, R., Foiani, M., Valsasnini, P., Badaracco, G., Cheriathundam, E. and Chang, L.M.S. (1985) J. Biol. Chem. 260, 7102–7107.Google Scholar
  30. 30.
    Scovassi, A.I., Stefanini, M. and Betrazzoni, U. (1984) J. Biol. Chem. 259, 10973–10977.Google Scholar
  31. 31.
    Chang, L.M.S., Plevani, R. and Bollum, F.J. (1982) J. Biol. Chem. 257, 5700–5706.Google Scholar
  32. 32.
    Rosenthal, A.L. and Lacks, S.A. (1977) Anal. Biochem. 80, 76–90.CrossRefGoogle Scholar
  33. 33.
    Hübscher, U., Pedrali-Noy, G., Krön, B.K., Deorfler, W. and Spadari, S. (1985) Anal. Biochem. 150, 442–448. 133CrossRefGoogle Scholar
  34. 34.
    Bowen, B., Steinberg, J., Laemmli, U.K. and Weintraub, H. (1980) Nucl. Acids Res. 8, 1–20.CrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1987

Authors and Affiliations

  • Robert P. Dottin
    • 1
  • Bodduluri Haribabu
    • 1
  • Clifford W. Schweinfest
    • 1
  • Richard E. Manrow
    • 1
  1. 1.Department of BiologyThe Johns Hopkins UniversityBaltimoreUSA

Personalised recommendations