Detection of Polypeptides on Immunoblots Using Enzyme-Conjugated or Radiolabeled Secondary Ligands
- 89 Downloads
Immunoblotting provides a simple and effective method for identifying specific antigens in a complex mixture of proteins. Initially, the constituent polypeptides are separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), or a similar technique, and are then transferred either electrophoretically or by diffusion onto a nitrocellulose filter. Once immobilized on a sheet of nitrocellulose, specific polypeptides can be identified using antibodies that bind to antigens retained on the filter and subsequent visualization of the resulting antibody-antigen complex. This chapter describes conditions suitable for binding antibodies to immobilized proteins and methods for locating these antibody-antigen complexes using appropriately labeled ligands. These methods are based on those of Blake et al. (1), Burnette (2) and Towbin et al. (3).
KeywordsMicrobial Contamination Nitrocellulose Filter Substrate Mixture Specific Polypeptide Diethanolamine Buffer
- 4.Tijssen, P. (1985) Practice and Theory of Enzyme Immunoassays. Elsevier, Amsterdam.Google Scholar
- 11.Langone, J. J. (1980) 125I-Labelled Protein A: Reactivity with IgG and use as a tracer in radioimmunoassay, in Methods in Enzymology Vol. 70 (Vunakis, H. V. and Langone, J. J., eds.), Academic Press New York, pp. 356–375.Google Scholar