Western Blot Hybridization

Authored by: Peter B. Kaufman , Ara Kirakosyan , Leland J. Cseke , E. Mitchell Seymour

Handbook of Molecular and Cellular Methods in Biology and Medicine

Print publication date:  December  2011
Online publication date:  April  2016

Print ISBN: 9781420069389
eBook ISBN: 9781439881958
Adobe ISBN:

10.1201/b11351-20

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Abstract

Western blot analysis is based on a protein–protein hybridization technique that is used for immune detection of specific antigen(s) of interest in a complex mixture of proteins. This is a simple, sensitive, and effective technology that has been used in immunology, molecular and cellular biology, and protein chemistry. The principle of western blotting is as follows: (1) A protein mixture is first separated according to molecular size using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). (2) The separated protein molecules are then immobilized onto a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. (3) The specific protein band of interest is identified by use of a specific antibody raised against a specific antigen (protein), which can specifically bind to the antigen (protein) of interest in the protein mixture that is immobilized on the membrane. (4) The antibody–antigen complex is then detected by an enzyme linked to a second antibody and substrate, or by the use of 125I-labeled protein A or its equivalent. 1 4 The antigen can also be directly detected with a fluorescence-labeled antibody, which directly binds to the antigen in a protein mixture. After washing away the non-bound antibody, the antigen-labeled antibody can then be visualized under a microscope fitted with a UV lamp that can excite the fluorescent tag using a specific wavelength of light. However, the direct method needs a relatively large amount of the labeled antibody to obtain good detection, and the labeling of an antibody of particular interest is expensive. Therefore, the indirect immunodetection method is widely used today. The following detailed protocols are based on modifications in the method of Towbin et al. 2 who first developed the technique.

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