Investigation of protein-tyro sine phosphatases by in-gel assays

Renaturation permits the detection of protein-tyrosine phosphatase (PTP) activities subsequent to separation by SDS-PAGE in the presence of the P-32-labeled poly(Glu(4)Tyr) as a macromolecular substrate. An efficient corresponding method has been developed by Burridge and Nelson [Anal. Biochem. 232 (1995) 56]. We describe here the modification of the basic method, its extension to two-dimensional gel electrophoresis, and applications to identify PTPs in signaling complexes and reversibly oxidized PTPs. Particular attention is given to the preparation of samples, to interpretation of the results as well as to advantages and limitations of the technique. Immunodepletion and the use of cells from knockout animals have been successful in the identification of individual PTPs. Readily detectable in cell lysates are PTP-PEST, SHP-2, SHP-1, PTP1B, and T-cell PTP. A much greater complexity of activity bands is, to a large extent, due to the generation of active fragments of PTPs. In-gel detection of PTPs can be employed to monitor cell fractionations and potential PTP activity changes. (C) 2004 Elsevier Inc. All rights reserved.