Recombinant Human Active SHP-2 Protein, CF
Recombinant Human Active SHP-2 Protein, CF Summary
Thr2-Arg593, with an N-terminal 6-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol and DTT.|
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Assay Buffer: 25 mM HEPES, 0.02% Brij-35, 1 mM DTT, pH 7.5
- Recombinant Human SHP-2 (rhSHP-2) (Catalog # 1894-SH)
- Substrate: Asp-Ala-Asp-Glu-Tyr(PO3)-Leu-Ile-Pro-Gln-Gln-Gly (Catalog # ES006)
- Malachite Green Phosphate Detection Kit (Catalog # DY996)
- 96 well Clear Plate (Costar, Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Prepare a Phosphate Standard curve by adding 10 µL of the 1 M Phosphate Standard to 990 µL of Assay Buffer for 10 mM stock. Continue by adding 10 µL of the 10 mM phosphate stock to 990 µL of Assay Buffer for 100 µM stock. (this is the first dilution to use as a standard).
- Perform six additional one-half dilutions of the 100 µM Phosphate stock.
- Load 50 µL of each standard dilution to empty wells of a clear plate. Include a curve blank containing 50 µL Assay Buffer. The standard curve has a range of 0.078-5 nmol per well.
- Dilute rhSHP-2 to 0.2 µg/mL in Assay Buffer.
- Dilute Substrate to 400 μM in Assay Buffer.
- Load 25 µL of 0.2 µg/mL rhSHP-2 to empty wells. For controls, add 25 µL of Assay Buffer.
- Start reaction by adding 25 µL of 400 μM Substrate to enzyme and control wells.
- Cover plate with plate sealer and incubate at 37 °C for 30 minutes.
- After incubation, add 30 µL of Malachite Green reagent A to all wells used. Mix briefly.
- Add 100 μL of deionized water to all wells. Mix briefly.
- Add 30 µL of Malachite Green reagent B to all wells. Mix and incubate at room temperature for 20 minutes.
- Read plate at 620 nm in endpoint mode.
- Calculate specific activity:
Specific Activity (μmol/min/mg) =
|Phosphate released* (nmol) x (0.001 μmol/1 nmol)|
|Incubation time (min) x amount of enzyme (mg)|
*Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for control.Per Reaction:
- rhSHP-2: 0.000005 mg
- Substrate: 200 μM
Src-Homology domain-2 containing protein tyrosine Phosphatase 2 (SHP-2), also called protein tyrosine phosphatase, non-receptor type 11 (PTPN11), PTP1D, PTP2C, and SYP, is an enzyme that dephosphorylates tyrosine residues in proteins. The protein contains two Src homology 2 (SH2) domains, which both regulate the activity of the enzyme (1) and allow it to selectively bind to SH2 sites on proteins such as Dok1, IRS1, and the insulin receptor (2). SHP-2 plays a unique stimulatory role in cell signaling. Cells lacking SHP-2 have poor mobility because the hyper-phosphorylation of FAK and other proteins in the focal adhesion complex (3) prevents turnover of cellular attachment points. Without SHP-2, sustained ERK stimulation does not take place (4). The Y992 phosphorylation site of EGFR is a particularly good substrate for SHP-2 (5) and a phosphopeptide containing this sequence can be used to measure the activity of the enzyme (Catalog # ES006) by detecting release of phosphate (Catalog # DY996).
- Zhao, Z. et al. (1994) J. Biol. Chem. 269:8780.
- Clemmons, D.R. and Maile, L.A. (2005) Mol. Endocrinol. 19:1.
- von Wichert, G. et al. (2003) EMBO J. 22:5023.
- Maroun, C.R. et al. (2000) Mol. Cell. Biol. 20:8513.
- Sugimoto, S. et al. (1993) J. Biol. Chem. 269:22771.
Citations for Recombinant Human Active SHP-2 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
Citations: Showing 1 - 2
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The tyrosine phosphatase SHP2 controls TGF?-induced STAT3 signaling to regulate fibroblast activation and fibrosis
Authors: A Zehender, J Huang, AH Györfi, AE Matei, T Trinh-Minh, X Xu, YN Li, CW Chen, J Lin, C Dees, C Beyer, K Gelse, ZY Zhang, C Bergmann, A Ramming, W Birchmeier, O Distler, G Schett, JHW Distler
Nat Commun, 2018;9(1):3259.
Sample Types: Recombinant Protein
Applications: Enzyme Assay
Sepsis-induced cardiac mitochondrial dysfunction involves altered mitochondrial-localization of tyrosine kinase Src and tyrosine phosphatase SHP2.
Authors: Zang Q, Martinez B, Yao X, Maass D, Ma L, Wolf S, Minei J
PLoS ONE, 2012;7(8):e43424.
Sample Types: Mitochondria
No product specific FAQs exist for this product, however you mayView all Proteins and Enzyme FAQs
Phosphatase Activity Assays
Phosphatase Peptide Substrates
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