Immunoaffinity purification of tyrosine-phosphorylated cellular proteins.
Transformation of cells by viral oncogene-encoded tyrosine kinases coincides with the phosphorylation of many cellular proteins on tyrosine. In order to study the potential cellular targets of oncogenic tyrosine kinases, tyrosine phosphoproteins were purified from cells by immunoaffinity chromatography with antibodies to phosphotyrosine. Tyrosine phosphoproteins were purified from both rat-1 cells and primary chicken embryo cells expressing transforming or non-transforming variants of the src oncogene. These proteins were released from anti-phosphotyrosine resins with hapten, and the protein mixtures contained 6-10 highly pure phosphoproteins including the src protein pp60src. The recovered proteins represented approximately 0.03% of total cellular proteins. All of the proteins were shown to contain phosphotyrosine; in addition, virtually all of these proteins were also phosphorylated on serine and threonine. This method thus provides a large-scale, single-step immunoaffinity purification of phosphotyrosine-containing proteins to a purity amenable for immunization protocols and characterization of individual polypeptides.
Kanner
S B
SB
Department of Microbiology, University of Virginia School of Medicine, Charlottesville 22908.
Reynolds
A B
AB
Parsons
J T
JT
D000818:Animals; D002645:Chickens; D002846:Chromatography, Affinity; D008970:Molecular Weight; D010449:Peptide Mapping; D010750:Phosphoproteins / Q000302:isolation & purification*; D019000:Phosphotyrosine; D011233:Precipitin Tests; D051381:Rats; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000276:immunology / Q000302:isolation & purification
D010750:Phosphoproteins; D019000:Phosphotyrosine; D014443:Tyrosine