Posttranslational phosphorylation of protein tyrosine residues in cells has been investigated by chemical and immunological methods to identify and characterize the catalysts and their substrates. Tyrosine phosphate (Tyr-P) residues are resistant to alkaline conditions (1 N NaOH, 65 degrees C) which destroy most Ser-P and Thr-P residues. A straightforward procedure to assay the base resistant [32-P]protein phosphoryl groups in in vitro labeled cells was developed by electroblotting SDS PAGE separated proteins to nylon blotting paper which can be incubated in base. This procedure is rapid and technically superior to treatment of gels. This technique was used to characterize the base resistant [32-P]phosphoproteins of several retrovirus transformed cell lines. Immunodecoration of proteins containing Tyr-P on electroblots is possible by incubating the electroblot of SDS gels with sheep antibodies which bind Tyr-P. The region of the blot with bound antibodies is detected with affinity purified anti.sheep IgG conjugated with horseradish peroxidase. The procedure was tested with authentic proteins containing Tyr-P or Ser-P residues and appears to be specific.
