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Shp-2 (human), (recombinant)

 
BML-SE273-0020 20 µg 404.00 USD
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Product Specification

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:Shipped on 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
 

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