UNIT 18.3 Phosphoamino Acid Analysis

  1. Bartholomew M. Sefton

Published Online: 1 MAY 2001

DOI: 10.1002/0471142727.mb1803s40

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Sefton, B. M. 2001. Phosphoamino Acid Analysis. Current Protocols in Molecular Biology. 40:18.3:18.3.1–18.3.8.

Author Information

  1. The Salk Institute, San Diego, California

Publication History

  1. Published Online: 1 MAY 2001
  2. Published Print: OCT 1997


It is often valuable to identify the phosphorylated residue in a protein. In the case of proteins phosphorylated at serine, threonine, or tyrosine, this is readily accomplished by partial acid hydrolysis in HCl followed by two-dimensional thin-layer electrophoresis of the labeled phosphoamino acid, as described here. Phosphothreonine andphosphotyrosine are more stable to hydrolysis in alkali than are RNA andpho sphoserine. Therefore, a protocol for mild alkaline hydrolysis of protein samples is also provided to enhance the detection of phosphothreonine and phosphotyrosine. Although this procedure can be carried out with a protein eluted from a preparative gel and concentrated by trichloroacetic acid or acetone precipitation, it is most easily accomplished by transfer of the protein of interest to a PVDF membrane.