Advertisement

Introduction to Protein Blotting

  • Biji T. Kurien
  • R. Hal Scofield
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 536)

Summary

Protein blotting is a powerful and important procedure for the immunodetection of proteins following electrophoresis, particularly proteins that are of low abundance. Since the inception of the protocol for protein transfer from an electrophoresed gel to a membrane in 1979, protein blotting has evolved greatly. The scientific community is now confronted with a variety of ways and means to carry out this transfer.

Key words

Western blotting Sodium dodecyl sulfate polyacrylamide gel electrophoresis Nitrocellulose membrane Polyvinylidene difluoride membrane 

References

  1. 1.
    Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to NC sheets: procedure and applications. Proc Natl Acad Sci USA 76, 4350–4354.PubMedCrossRefGoogle Scholar
  2. 2.
    LeGendre, N. (1990). Immobilon-P transfer membrane: applications and utility in protein biochemical analysis. Biotechniques 9 (6 Suppl), 788–805. Review.PubMedGoogle Scholar
  3. 3.
    Southern, E.M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98, 503–517.PubMedCrossRefGoogle Scholar
  4. 4.
    Alwine, J. C., Kemp, D. J., Stark, G.R. (1977) Method for detection of specific RNAs in agar gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci USA 74, 5350–5354.PubMedCrossRefGoogle Scholar
  5. 5.
    Burnette, W.N. (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate–polyacrylamide gels to unmodified NC and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112, 195–203.PubMedCrossRefGoogle Scholar
  6. 6.
    Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  7. 7.
    Kost, J., Liu, L-S., Ferreira, J., and Langer, R. (1994) Enhanced protein blotting from PhastGel media to membranes by irradiation of low-intensity. Anal Biochem 216, 27–32.PubMedCrossRefGoogle Scholar
  8. 8.
    Gershoni, J.M. and Palade, G.E. (1982) Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to a positively charged membrane filter. Anal Biochem 124, 396–405.PubMedCrossRefGoogle Scholar
  9. 9.
    Gershoni, J.M. (1988) Protein blotting: a manual. Methods Biochem Anal33, 1–58. Review.PubMedCrossRefGoogle Scholar
  10. 10.
    Kurien, B.T. and Scofield, R.H. (2006) Western blotting. Methods 38, 283–293.PubMedCrossRefGoogle Scholar
  11. 11.
    Karey, K.P. and Sirbasku, D.A. (1989) Glutaraldehyde fixation increases retention of low molecular weight proteins (growth factors) transferred to nylon membranes for western blot analysis. Anal Biochem 178, 255–259.PubMedCrossRefGoogle Scholar
  12. 12.
    Harlow, E. and Lane, D. (1988) Immunoblotting. In: Antibodies. A Laboratory Manual, Harlow, E. and Lane, D. (eds.). Cold Spring Harbor Laboratory, New York, p. 485.Google Scholar
  13. 13.
    Renart, J., Reiser, J., and Stark, G.R. (1979) Transfer of proteins from gels to diazobenzyloxymethyl paper and detection with anti-sera: a method for studying antibody specificity and antigen structure. Proc Natl Acad Sci USA, 76, 3116–3120.PubMedCrossRefGoogle Scholar
  14. 14.
    Elkon, K.B., Jankowski, P.W., and Chu, J.L. (1984) Blotting intact immunoglobulins and other high-molecular-weight proteins after composite agarose-polyacrylamide gel electrophoresis. Anal Biochem 140, 208–213.PubMedCrossRefGoogle Scholar
  15. 15.
    Gibson, W. (1981). Protease-facilitated transfer of high-molecular-weight proteins during electrotransfer to NC. Anal Biochem 118, 1–3.PubMedCrossRefGoogle Scholar
  16. 16.
    Bolt, M.W. and Mahoney, P.A. (1997) High efficiency blotting of proteins of diversesizes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem 247, 185–192.PubMedCrossRefGoogle Scholar
  17. 17.
    Kurien, B.T. and Scofield, R.H. (2002) Heat mediated, ultra-rapid electrophoretic transfer of high and low molecular weight proteins to NC membranes. J Immunol Methods 266, 127–133.PubMedCrossRefGoogle Scholar
  18. 18.
    Gershoni, J.M. and Palade, G.E. (1983) Protein blotting: principles and applications. Anal Biochem 131, 1–15.PubMedCrossRefGoogle Scholar
  19. 19.
    Thornton, D.J., Carlstedt, I., and Sheehan, J.K. (1996) Identification of glycoproteins on nitrocellulose membranes and gels. Mol Biotechnol 5, 171–176.PubMedCrossRefGoogle Scholar
  20. 20.
    Tonkinson, J.L. and Stillman, B. (2002) NC: a tried and true polymer finds utility as a post-genomic substrate. Front Biosci 7, c1–c12. Review.PubMedCrossRefGoogle Scholar
  21. 21.
    Lauritzen, E., Masson, M., Rubin, I., ­Bjerrum, O.J., and Holm, A. (1993) Peptide dot immunoassay and immunoblotting: electroblotting from aluminum thin-layer chromatography plates and isoelectric focusing gels to activated NC. Electrophoresis 14, 852–859.PubMedCrossRefGoogle Scholar
  22. 22.
    Masson, M., Lauritzen, E., and Holm, A. (1993) Chemical activation of NC membranes for peptide antigen–antibody binding studies: direct substitution of the nitrate group with diaminoalkane. Electrophoresis 14, 860–865.PubMedCrossRefGoogle Scholar
  23. 23.
    Too, C.K., Murphy, P.R., and Croll, R.P. (1994) Western blotting of formaldehyde-fixed neuropeptides as small as 400 daltons on gelatin-coated NC paper. Anal Biochem 219, 341–348.PubMedCrossRefGoogle Scholar
  24. 24.
    Coull, J.M., Dixon, J.D., Laursen, R.A., Koester, H., and Pappin, D.J.C. (1989) Development of membrane supports for the solid-phase sequence analysis of proteins and peptides. In: Methods in Protein Sequence Analysis, Witmann-Liebold, B. (ed.). Springer, Berlin, pp. 69–78.CrossRefGoogle Scholar
  25. 25.
    Matsudaira, P. (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262,10035–10038.PubMedGoogle Scholar
  26. 26.
    Pluskal, M.F., Przekop, M.B., Kavonian, M.R., Vecoli, C., and Hick, D.A. (1986) Biotechniques 4, 272–282.Google Scholar
  27. 27.
    Kittler, J.M., Meisler, N.T., Viceps-Madore, D., Cidlowski, J.A., Thanassi, J.W. (1984) A general immunochemical method for detecting proteins on blots. Anal Biochem 137, 210–216.PubMedCrossRefGoogle Scholar
  28. 28.
    Hughes, J.H., Mack, K., and Hamparian, V.V. (1988) India ink staining of proteins on nylon and hydrophobic membranes. Anal Biochem 173, 18–25.PubMedCrossRefGoogle Scholar
  29. 29.
    Tovey, E.R. and Baldo, B.A. (1989) Protein binding to NC, nylon and PVDF membranes in immunoassays and electroblotting. J Biochem Biophys Methods 19, 169–183.PubMedCrossRefGoogle Scholar
  30. 30.
    Xu, Q.Y. and Shively, J.E. (1988) Microsequence analysis of peptides and proteins. VIII. Improved electroblotting of proteins onto membranes and derivatized glass-fiber sheets. Anal Biochem 170, 19–30.PubMedCrossRefGoogle Scholar
  31. 31.
    Nielsen, P.J. (1982) The phosphorylation of ribosomal protein S6 in rat tissues following cycloheximide injection, in diabetes, and after denervation of diaphragm. A simple immunological determination of the extent of S6 phosphorylation on protein blots. J Biol Chem 257, 12316–12321.PubMedGoogle Scholar
  32. 32.
    Kurien, B.T. and Scofield, R.H. (1997) Multiple immunoblots after non-electrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens. J Immunol Methods 205, 91–94.PubMedCrossRefGoogle Scholar
  33. 33.
    Kyhse-Andersen, J. (1984) Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 10, 203–209.PubMedCrossRefGoogle Scholar
  34. 34.
    Otter, T., King, S.M., and Witman, G.B. (1987) A two-step procedure for efficient electro transfer of both high-molecular weight (greater than 400,000) and low-molecular weight (less than 20,000) proteins. Anal Biochem 162, 370–377.PubMedCrossRefGoogle Scholar
  35. 35.
    Harper, D.R., Kit, M.L., and Kangro, H.O. (1990) Protein blotting: ten years on. J Virol Methods 30, 25–39. Review.PubMedCrossRefGoogle Scholar
  36. 36.
    Egger, D. and Bienz, K. (1994) Protein (western) blotting. Mol Biotechnol 1, 289–305.PubMedCrossRefGoogle Scholar
  37. 37.
    Wisdom, G.B. (1994) Protein blotting. Methods Mol Biol 32, 207–213.PubMedGoogle Scholar
  38. 38.
    Kurien, B.T. and Scofield, R.H. (2003) Protein blotting: a review. J Immunol Methods 274, 1–15. Review.PubMedCrossRefGoogle Scholar
  39. 39.
    Kurien, B.T. and Scofield, R.H. (2005) Blotting techniques, In: Encyclopedia of Analytical Science, Second Edition (Worsfold, P.J., Townshend, A., and Poole, C.F., eds.). Elsevier, Oxford, p. 425.Google Scholar
  40. 40.
    Reinhart, M.P. and Malamud, D. (1982) Protein transfer from isoelectric focusing Gels: the native blot. Anal Biochem 123, 229–235.PubMedCrossRefGoogle Scholar
  41. 41.
    Jagersten, C., Edstrom, A., Olsson, B., and Jacobson, G. (1988) Blotting from PhastGel media after horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresis 9, 662–665.PubMedCrossRefGoogle Scholar
  42. 42.
    Kazemi, M. and Finkelstein R.A. (1990) Checkerboard immunoblotting (CBIB): an efficient, rapid, and sensitive method of assaying multiple antigen/antibody cross-reactivities. J Immunol Methods 128, 143–146.PubMedCrossRefGoogle Scholar
  43. 43.
    Heukeshoven, J. and Dernick, R. (1995). Effective blotting of ultrathin polyacrylamide gels anchored to a solid matrix. Electrophoresis 16, 748–756.PubMedCrossRefGoogle Scholar
  44. 44.
    Olsen, I. and Wiker, H.G. (1998) Diffusion blotting for rapid production of multiple identical imprints from sodium dodecyl sulfate polyacrylamide gel electrophoresis on a solid support. J Immunol Methods 220, 77–84.PubMedCrossRefGoogle Scholar
  45. 45.
    Chen, H. and Chang, G.D. (2001) Simultaneous immunoblotting analysis with activity gel electrophoresis in a single polyacrylamide gel. Electrophoresis 22, 1894–1899.PubMedCrossRefGoogle Scholar
  46. 46.
    Bowen, B., Steinberg, J., Laemmli, U.K., and Weintraub, H. (1980) The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res 8, 1–20.PubMedCrossRefGoogle Scholar
  47. 47.
    Kurien, B.T. and Scofield, R.H. (2000) Association of neutropenia in systemic lupus erythematosus with anti-Ro and binding of an immunologically cross-reactive neutrophil membrane antigen. Clin Exp Immunol 120, 209–217.PubMedCrossRefGoogle Scholar
  48. 48.
    Kurien, B.T., Matsumoto, H., and Scofield, R.H. (2001) Purification of tryptic peptides for mass spectrometry using polyvinylidene fluoride membrane. Indian J Biochem Biophys 38, 274–276.PubMedGoogle Scholar
  49. 49.
    Bischoff, K.M., Shi, L., and Kennelly, P.J. (1998) The detection of enzyme activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem 260, 1–17. Review.PubMedCrossRefGoogle Scholar
  50. 50.
    Peferoen, M., Huybrechts, R., and De Loof, A. (1982) Vacuum-blotting: a new simple and efficient transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to NC. FEBS Lett 145, 369–372.CrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Oklahoma Medical Research FoundationOklahoma CityUSA

Personalised recommendations