Transfer Techniques in Protein Blotting
Polyacrylamide gel electrophoresis is an extremely powerful tool for the analysis of complex protein mixtures. Although the value of this method cannot be questioned, it is restricted in that the separated proteins remain buried within the dense gel matrix and are not readily available for further investigation. A number of methods have been developed in order to try and overcome this problem, for example the elution of proteins from excised gel slices (see Chapter 19). Alternatively, proteins have been studied while they are still buried within the gel using a variety of in situ peptide mapping (see Chapter 22) and gel overlay techniques (for example, see ref. 1). Unfortunately all of these methods have serious drawbacks: in the case of protein elution and in situ peptide mapping techniques, the resolution and number of bands that can be processed is restricted, whereas the gel overlay techniques are generally time-consuming and insensitive.
KeywordsEquilibration Buffer Nitrocellulose Filter Transfer Buffer High Molecular Weight Protein Equilibration Step
- 2.Gooderham, K. (1983) Protein blotting. In Techniques in molecular biology, Walker J. M., and Gaastra, W., eds. pp 49–61. Croom Helm Publishers.Google Scholar
- 6.Tsang, V. C. W., Peralta, J. M., and Simons, A. R. (1983) Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. In Methods in Enzymology 92, Langone, J. J. and Van Vunakis, H. V. eds. pp. 377–391. Academic Press, New York.Google Scholar
- 8.Jack, R. S., Brown, M. T., and Gehring, W. J. (1983) Protein blotting as a means to detect sequence-specific DNA-binding proteins. Cold Spring Harbor Symp. Quant. Bid., XLV11, 483–491.Google Scholar