The PY20 antibody against phosphotyrosine is useful for studying the mechanism of action of phosphotyrosine kinases using the method of 2D gel Western blotting (WB). Differences between control and treated samples may easily be found on 2D gels because the WB patterns are much simpler than the very complex patterns obtained from conventional staining. If the PVDF blot is stained with Coomassie blue and scanned prior to immunostaining, matching between the ECL film and the Coomassie pattern of a duplicate 2D gel becomes straightforward. Proteins of interest may then be cut from the duplicate Coomassie gel and identified by mass spectrometry (MS).

However, in many cases these important proteins are of too low an abundance to be identified from the original sample. In this paper we describe a method for immunoprecipitation of phosphotryosine-containing proteins from large amounts of sample prepared for 2D electrophoresis, in order to get enough material for MS. Approaches, problems and results will be presented.