Current Protocols in Immunology
DOI: 10.1002/0471142735.im1107s93
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Tyrosine phosphorylation and dephosphorylation of proteins play a critical role during many processes of the immune system, from early development to fully differentiated effector function. Since the opposing actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) determine the steady state level of tyrosine phosphorylation on a given protein, it is often important for mechanistic studies to determine the specific activities of PTKs and PTPs. PTPs are defined by their enzymatic activity that catalyzes the dephosphorylation of phosphotyrosine residues. This unit focuses on methods to determine the enzymatic activity of PTPs. While there are many varieties of PTP assays, the focus in this unit is on immune complex PTP assays, which do not require elaborate biochemical purifications and are commonly used to test the activities of specific PTPs in the immune system. Although it has become increasingly recognized that PTPs show substrate specificities, most in vitro PTP assay are based on the use of generic surrogate substrates. In the basic protocol provided in this unit, dephosphorylation of the chromogenic substrate p-Nitrophenyl Phosphate (pNPP) is measured to determine the enzymatic activity of the PTP. In the first alternative protocol, the release of inorganic phosphate from a phosphopeptide is quantified using the Malachite Green Assay. The second alternative protocol uses a radioactively-labeled phosphoprotein as substrate, and the amount of released radioactivity is used to assess PTP activity.