| BML-SE273-0020 | 20 µg | 531.00 USD |  |
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| Alternative Name: | Src homology-2 containing phosphatase 2 |
| MW: | 68 kDa |
| Source: | Produced in E. coli. full length human Shp-2 (aa 1-593). |
| UniProt ID: | Q06124 |
| Formulation: | Liquid. In 10mM MES, pH 5.7, containing 100mM NaCl, 5mM DTT and 33% glycerol. |
| Purity: | ≥90% (SDS-PAGE) |
| Purity Detail: | Purified by multi-step chromatography. |
| Specific Activity: | ≥100pmol/min/µg assayed by p-nitrophenylphosphate (pNPP, 50mM) hydrolysis at pH 7.0, 30°C. |
| Shipping: | Dry Ice |
| Long Term Storage: | -80°C |
| Use/Stability: | After initial defrosting, aliquot product into individual tubes and snap freeze, for example in liquid nitrogen or dry ice/ethanol. |
| Handling: | Avoid freeze/thaw cycles. |
| Scientific Background: | Shp-2 is one of two human Src Homology-2 (SH2) containing phosphatases, enzymes in which two regulatory phosphotyrosine binding domains (N-SH2 & C-SH2) lie N-terminal to the catalytic (PTP) domain. In most cases, full activation of the Erk/MAP kinase pathway by receptor tyrosine kinase signaling requires Shp-2, which is thought to act somewhere upstream of Ras. |
| Protocol: | PNPP Assay for Shp-2 (BML-SE273) Assay Buffer: 25mM Tris, pH 7.0, 50mM NaCl, 2mM EDTA, 5mM DTT, 0.01% Brij35, 1mg/ml BSA. Store on ice bath. p-nitrophenylphosphate (pNPP) stock solution: 100mM pNPP (sodium salt) dissolved in assay buffer [PROTECT FROM LIGHT!] Store at RT. Shp-2 (BML-SE273): Dilute ten-fold in Assay Buffer to 0.1mg/ml and store on ice. ASSAY IN 96-WELL ½-VOLUME MICROPLATE 1. Add 45μl assay buffer to the desired number of wells of the microplate and warm to 30°C. 2. Add 5μl Shp-2 (0.1mg/ml; stored on ice) to the 45μl of Assay Buffer. 3. Start reaction by adding 50μl pNPP stock solution, prewarmed to 30°C. Mix thoroughly. Final pNPP concentration=50mM. 4. Follow A405nm in a plate-reading visible spectrophotometer. Monitor for 5-20 min., recording, for example, A405nm at 1 min. intervals. DATA ANALYSIS 1. Plot A405nm versus time. Determine slope (OD/min.) in linear part of curve. 2. At the pH of the assay (7.0) the extinction coefficient (ε) for p-nitrophenol is ~18,000 OD M-1 cm-1. The optical pathlength for 100μl in a ½-volume 96-well microplate is 0.5cm. The rate of phosphate release in pmol/min would be: Rate of hydrolysis (pmol/min.) = Slope (OD/min.) x (100 x 10-6 L x 1012 pmol/mol)/(18,000 OD L mol-1 cm-1 x 0.5 cm) = Slope (OD/min.) x 24,000 pmol/OD Specific Activity = x pmol/min ÷ total amount of protein |
| Regulatory Status: | RUO - Research Use Only |
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