EGF Receptor Substrate 2 Phospho-Tyr5

Kinase experiment:

Briefly, the assays are performed in 50 μL volumes in a microtiter plate using the generalized PTPase substrates ENDpYINASL and DADEpYLIPQQG (where pY represents phosphotyrosine) and assay buffer containing 25 mM Tris-HCl (pH 7.4), 50 mM NaCl, 2 mM EDTA, 5 mM dithiothreitol, 0.01% Brij 35, and 1 mg of bovine serum albumin/mL. The reactions are started by the addition of 5 μg of either CAV GST-VP2, GST, CAV GST-VP2 containing the C95S mutation, or TLMV GST-ORF2 or 2 units of the positive control T cell protein-tyrosine phosphatase (TC-PTP) in assay buffer. Control reactions are assayed with either ENDpYINASL substrate alone, DADEpYLIPQQG substrate alone, CAV GST-VP2 without substrate, CAV GST-VP2 containing the C95S mutation without substrate, TLMV GST-ORF2 without substrate, TC-PTP without substrate, or assay buffer with neither enzyme nor substrate. A phosphate standard curve is derived using a supplied phosphate standard. The reactions are terminated by the addition of the malachite green detection reagent.

References:

[1]. Peters MA, et al. Chicken anemia virus VP2 is a novel dual specificity protein phosphatase. J Biol Chem. 2002 Oct 18;277(42):39566-73. Epub 2002 Jul 31.
[2]. Wu L, et al. Comparative kinetic analysis and substrate specificity of the tandem catalytic domains of the receptor-like protein-tyrosine phosphatase alpha. J Biol Chem. 1997 Mar 14;272(11):6994-7002.