Unit

UNIT 11.3 Antiphosphotyrosine Blotting

  1. Jeffrey N. Siegel

Published Online: 1 MAY 2001

DOI: 10.1002/0471142735.im1103s03

Current Protocols in Immunology

Current Protocols in Immunology

How to Cite

Siegel, J. N. 2001. Antiphosphotyrosine Blotting. Current Protocols in Immunology. 3:11.3:11.3.1–11.3.7.

Author Information

  1. Naval Medical Research Institute, Bethesda, Maryland

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: SEP 1992

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Abstract

Antiphosphotyrosine blotting is a technique for detecting tyrosine-phosphorylated substrates by the use of antibodies that recognize these residues on a wide variety of proteins. This unit describes conditions for cell lysis and immunoprecipitation of proteins with an antiphosphotyrosine antibody, followed by electrophoretic separation, immunoblotting, and color detection of the blotted proteins. This combination of steps provides particularly sensitive conditions for the detection of tyrosine-phosphorylated substrates, but also gives good results for any protein transferred to nitrocellulose, including whole-cell lysates or proteins immunoprecipitated with another antibody. Although the alkaline phosphatase color-detection system has the advantage of providing superior resolution and higher sensitivity without the use of any radioactivity, the 125I-labeled Staphylococcus protein A detection system is described for use in conjunction with the blotting protocol.