UNIT 18.3 Phosphoamino Acid Analysis
Published Online: 1 MAY 2001
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Molecular Biology
How to Cite
Sefton, B. M. 2001. Phosphoamino Acid Analysis. Current Protocols in Molecular Biology. 40:18.3.1–18.3.8.
- Published Online: 1 MAY 2001
- 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.