| Assay Method Information | |
| | PTP Assay |
| Description: | PTP assays were conducted as previously described in Kontaridis et al., J Biol Chem. 2006; 281:6785-6792, using para-nitrophenyl phosphate (pNPP, obtained from Sigma) as substrate. Briefly, WT C57/B16, MRL/MpJ, or MRL/lpr tissue (spleen, kidney, heart) lysates were homogenized and lysed in RIPA buffer (but without sodium orthovanadate), and SHP2 was immunoprecipitated by using anti-SHP2 polyclonal antibodies (Santa Cruz Biotechnology Inc.) coupled to protein A-Sepharose Immune complexes were washed 3 times in RIPA buffer without sodium orthovanadate and once in wash buffer [30 mM HEPES (pH 7.4), 120 mM NaCl without pNPP]. For each sample, PTP assays were performed in triplicate at 37° C. in 50 μl of assay buffer [30 mM Hepes (pH 7.4), 120 mM NaCl, 5 mM dithiothreitol, 10 mM pNPP] containing 50 μl of the SHP2 beads. Reactions were terminated with 0.2 N NaOH and phosphate release was determined by measuring A410. Following the assays, immune complexes were recovered by centrifugation, boiled in 2× SDS-PAGE sample buffer, resolved by SDS-PAGE, and immunoblotted with polyclonal SHP2 antibodies (Santa Cruz Biotechnology Inc.) to ensure that equal amounts of SHP2 had been tested for phosphatase activity. |
| Affinity data for this assay | |
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