Question (20575) | Recombinant human SHP2 protein (Active) (ab42578)

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Question

ab42578 SHP2 protein (Tagged) ab48657 SHP1 protein (Human) I didn't detect any activity - no color change in PTP assay. Can you provide the protocol used to determine the protein activity?

Answer

Thank you for your patience in this matter. Regarding ab48657 (SHP1 protein (Human)), the originator has informed us that they define one unit as the amount of enzyme that will hydrolyze 1nmole of p-nitrophenyl phosphate(pNPP, Sigma N4645 ) per minute at 37C, pH7.4 using 10mM of substrate(pNPP). After receiving your enquiry, they re-tested the product and found that SHP-1 is functional (phosphatase activity detected). I have attached the protocol that they have used to determine the phosphatase activity of SHP-1. In regards to ab42578 (SHP2 protein (Tagged)), the originator has provided the following protocol: SHP-2 Assay Materials Assay buffer: 50 mM HEPES, pH 7.4, 100 Mm NaCl, 2 mM EDTA, 3 mM DTT. Prepare 5 X pNPP substrate (10 mM) in the assay buffer Enzyme preparation: SHP-2 (1 mg/ml), dilute 1:25 in the assay buffer Enzymatic Reactions by pNPP hydrolysis 1. Prepare reaction mixtures in a 96-well plate (keep on ice). Add in the order: 60 ul of assay buffer 20 ul of 10 mM pNPP in buffer (final conc. 2 mM) 10 ul 1% BSA 10 ul of 40 ng/ul SHP-2 enzyme in assay buffer Mix well and start reaction at 30C water bath, and incubate for 10 min. 2. Add 100 ul per well of 2 M K2CO3 to stop the reaction. 3. Read absorbance at 405 nm using a plate reader. I hope this information helps.

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