Reutilization of Western Blots After Chemiluminescent Detection or Autoradiography
Western blotting (also called immunoblotting) is a widely utilized laboratory procedure that involves formation and detection of antibody-antigen complexes between antibodies that are initially in solution and antigens that are immobilized on derivatized paper (reviewed in refs. 1-3). This procedure is most commonly performed by sequentially subjecting a complex mixture of polypeptides to three manipulations: (1) electrophoretic separation, usually through polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS); (2) electrophoretic transfer of the separated polypeptides to thin sheets of nitrocellulose or polyvinylidene fluoride (PVDF); and (3) reaction of the sheets sequentially with one or more antibody-containing solutions. Because these are laborious, low-throughput procedures, there has been considerable interest over the past 15 yr in increasing the information gained from immunoblots in various ways. The approaches to this problem have included removal of bound antibodies so that immobilized polypeptides can be reprobed with additional antisera (last reviewed in ref. 4), the sequential (5) or simultaneous (6) use of multiple antisera to detect different antigens, and the development of higher density filter manifolds to permit more spots to be placed in the same area when purified proteins are applied to filters by spot adsorption. After a brief review of critical parameters for successful immunoblotting, some of these approaches are briefly discussed and illustrated.
KeywordsUrea Glycine Electrophoresis Penicillin Immobilization
- 4.Kaufmann, S. H. and Kellner, U. (1998) Erasure of Western blots after autoradiographic or chemiluminescent detection, in Methods in Molecular Biology, Vol. 80, Humana Press, Totowa, NJ, pp. 223–235.Google Scholar
- 11.Hashida, S., Hashinaka, K., Ishikawa, S., and Ishikawa, E. (1997) More reliable diagnosis of infection with human immunodeficiency virus type 1 (HIV-1) by detection of antibody IgGs to Pol and Gag proteins of HIV-1 and p24 antigen of HIV-1 in urine, saliva, and/or serum with highly sensitive and specific enzyme immunoassay (immune complex transfer enzyme immunoassay): a review. J. Clin. Lab. Analyt. 11, 267–286.CrossRefGoogle Scholar
- 13.Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Google Scholar
- 19.Gallagher, S. R. (1999) One-dimensional SDS gel electrophoresis of proteins, in Current Protocols in Molecular Biology, Vol. 2, John Wiley & Sons, New York, pp. 10.2A.1–10.2A.34.Google Scholar