Antibodies, A Laboratory Manual, Second Edition, Edited by Edward A. Greenfield



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Blocking and Incubation with Antibodies of Immunoblots Prepared with Immunoprecipitated Protein Antigens (Immunoprecipitation/Western Blotting)

(Protocol summary only for purposes of this preview site)

Detection of the protein antigens in immunoblots prepared with immunoprecipitated protein antigens can be affected by the presence of high amounts of the immunoprecipitating antibodies. When it is not possible to use the immunoprecipitating antibodies and the primary antibodies raised in different species, the following protocol provides a convenient and inexpensive alternative to achieve optimal detection of immunoprecipitated protein antigens. In this protocol, a nitrocellulose or PVDF membrane containing immunoprecipitated protein samples is rinsed with ultrapure H2O after the transfer of proteins and detection of the total proteins using Ponceau S dye (optional; see Protocol 7). Blocking solution is applied to the membrane, and the membrane is incubated and then rinsed off (optional) before addition of the primary antibody labeled with biotin. After washing, the membrane is incubated with enzyme- or fluorochrome-labeled avidin for detection.

Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition

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