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Biology

Immunoblot Analysis

doi: 10.3791/759 Published: June 20, 2008

Summary

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. This video provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Abstract

Immunoblotting (western blotting) is a rapid and sensitive assay for the detection and characterization of proteins that works by exploiting the specificity inherent in antigen-antibody recognition. It involves the solubilization and electrophoretic separation of proteins, glycoproteins, or lipopolysaccharides by gel electrophoresis, followed by quantitative transfer and irreversible binding to nitrocellulose, PVDF, or nylon. The immunoblotting technique has been useful in identifying specific antigens recognized by polyclonal or monoclonal antibodies and is highly sensitive (1 ng of antigen can be detected). This unit provides protocols for protein separation, blotting proteins onto membranes, immunoprobing, and visualization using chromogenic or chemiluminescent substrates.

Protocol

The complete text protocol for this experimental approach is available in Current Protocols in Molecular Biology

Disclosures

The authors have nothing to disclose.

Immunoblot Analysis
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Cite this Article

Gallagher, S., Chakavarti, D. Immunoblot Analysis. J. Vis. Exp. (16), e759, doi:10.3791/759 (2008).More

Gallagher, S., Chakavarti, D. Immunoblot Analysis. J. Vis. Exp. (16), e759, doi:10.3791/759 (2008).

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