Cell or tissue lysates were subjected to immunoprecipitation (IP) with antibody against SHP-2 (BD Transduction Laboratories). The immunocomplex was pooled down by protein A/G agarose (Thermo Scientific, Schwerte, Germany) beads. SHP-2 PTP activity was measured using Tyrosine Phosphatase Assay Kit (Promega, Madison, WI, USA) as specified by its manufacturer.
Tyrosine phosphatase assay kit
Manufactured by Promega
Sourced in United States
The Tyrosine Phosphatase Assay Kit is a laboratory tool used to measure the activity of tyrosine phosphatase enzymes. It provides a quantitative colorimetric method to determine the level of phosphatase activity in a sample.
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4 protocols using tyrosine phosphatase assay kit
1
SHP-2 Phosphatase Activity Assay
2
Tyrosine Phosphatase Activity Assay
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The non-radioactive Tyrosine Phosphatase Assay System in vitro was performed with the tyrosine phosphatase assay kit (Promega Corporation, Madison, WI, USA) using Tyr phosphopeptide (END(pY)INASL), where pY represents phosphotyrosine as substrate. 96-well dishes containing 0, 100, 200, 500, 1,000 and 2,000 pmol free phosphate and 50 µl of reaction buffer were prepared for a standard curve. Precipitates were washed 3 times in lysis buffer and were subjected to 50 µl of phosphatase buffer (0.9% NaCl, 0.1 mg/ml BSA, 20 mM imidazole, pH 7.2) [21] (link). Immunoprecipitated PTPRT (10 µl) was then added to reaction buffer as purified enzyme preparations. Phosphatase activity was shown as the level of phosphate released (pmol/min/µl).
3
Measuring SHP-2 Phosphatase Activity
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After treatment of TA, U2OS or xenograft tumor tissue protein extract were incubated with anti-SHP-2 antibody (Ab) in immunoprecipitation (IP) buffer (20 mM of Tris-HCl, 150 mM of NaCl, 1 mM of ethylenediaminetetraacetic acid, 1 mM DTT, 1% beta-mercaptoethanol, 1% TritonX-100, 1 mM MgCl2 and 0.1% BSA, 1% Protease inhibitor, pH 7.5) overnight. Protein A/G-Agarose Resin (Yeasen, Shanghai, China) was added to each group, followed by incubation for 3 h at 4 °C with rotation. After centrifugation, SHP-2 PTP activity was measured using the Tyrosine Phosphatase Assay Kit (Promega, Madison, WI, USA) as specified by its manufacturer. The purified recombinant SHP-2 proteins (Novus Biologicals, Littleton, CO, USA) were preincubated with different concentrations of TA in the reaction mixture. The detection method of purified SHP-2 PTP activity was as same as the IP samples.
4
Biochemical Characterization of PTPσ Catalytic Domains
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The two intracellular catalytic domains (D1 and D2) of Ptprs were cloned into a pET28a vector, overexpressed in Escherichia coli BL21 and purified65 (link). Enzymatic activity of PTPσ was assayed using a modified version of the Malachite Green Assay66 (link) and the Tyrosine Phosphatase Assay Kit (Promega Corporation). Unless stated otherwise, standard assays were carried out using 50 nM PTPσ protein in 1× Buffer (10 mM Tris, 5 mM MgCl2, 10 mM NaCl, 0.02% Tween) and Tyr Phosphopeptide as substrate (200 μM for Fig. 1b, c , 50–1200 μM in Fig. 1c ). Purified catalytic domain 1 and domain 2 of PTPσ were pre-incubated with test compounds 3071, 5205, 5075, or control for 15 min in a 96-well plate before the addition of Tyr Phosphopeptide (DADE(pY)LIPQQG). For IC50 determination, rates normalized relative to uninhibited controls (dimethyl sulfoxide (DMSO)) were plotted against compound concentration and fitted using a four-parameter nonlinear regression curve fit ((y = [(A − D) (1 + {xC − 1}B) − 1] + D) (Prism 6.0, Graphpad Software). For mechanism studies and determination of the enyzme’s Km and Vmax, data were analyzed using a nonlinear regression fit according to the classical Michaelis–Menten kinetics model, Y = Vmax*X/(Km + X) (Prism 6.0, Graphpad Software).
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