Recombinant Human Active SHP-2 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Active SHP-2 Protein, CF Summary

• Details of Functionality: Measured by its ability to dephosphorylate a tyrosine residue in a peptide containing the EGFR Y992 phosphorylation site (Catalog # ES006). The specific activity is >3 µmol/min/mg, measured under the described conditions.
• Source: E. coli-derived human SHP-2 protein
  Thr2-Arg593, with an N-terminal 6-His tag
• Accession #: NP_002825.3
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: PTPN11
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 69 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 63-70 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol and DTT.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • 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
  1. 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).
  2. Perform six additional one-half dilutions of the 100 µM Phosphate stock.
  3. 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.
  4. Dilute rhSHP-2 to 0.2 µg/mL in Assay Buffer.
  5. Dilute Substrate to 400 μM in Assay Buffer.
  6. Load 25 µL of 0.2 µg/mL rhSHP-2 to empty wells. For controls, add 25 µL of Assay Buffer.
  7. Start reaction by adding 25 µL of 400 μM Substrate to enzyme and control wells.
  8. Cover plate with plate sealer and incubate at 37 °C for 30 minutes.
  9. After incubation, add 30 µL of Malachite Green reagent A to all wells used. Mix briefly.
  10. Add 100 μL of deionized water to all wells. Mix briefly.
  11. Add 30 µL of Malachite Green reagent B to all wells. Mix and incubate at room temperature for 20 minutes.
  12. Read plate at 620 nm in endpoint mode.
  13. 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Active SHP-2 Protein, CF

• BPTP3
• EC 3.1.3.48
• MGC14433
• protein tyrosine phosphatase, non-receptor type 11
• protein tyrosine phosphatase-2
• Protein-tyrosine phosphatase 1D
• Protein-tyrosine phosphatase 2C
• PTP1D
• PTP-1D
• PTP2C
• PTP-2C
• PTP2CNS1
• PTPN11
• SHP2
• SHP-2
• SHP2CFC
• SHPTP2
• SH-PTP2
• SH-PTP2Noonan syndrome 1
• SH-PTP3
• tyrosine-protein phosphatase non-receptor type 11

Background

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).

1. Zhao, Z. et al. (1994) J. Biol. Chem. 269:8780.
2. Clemmons, D.R. and Maile, L.A. (2005) Mol. Endocrinol. 19:1.
3. von Wichert, G. et al. (2003) EMBO J. 22:5023.
4. Maroun, C.R. et al. (2000) Mol. Cell. Biol. 20:8513.
5. Sugimoto, S. et al. (1993) J. Biol. Chem. 269:22771.

Publications for SHP-2/PTPN11 (1894-SH)(5)

Publications using 1894-SH

• Liu, WS;Zhao, JF;Guo, XJ;Lu, SZ;Li, W;Li, WZ; Design, synthesis, activity and molecular dynamics studies of 1,3,4-thiadiazole derivatives as selective allosteric inhibitors of SHP2 for the treatment of cancer European journal of medicinal chemistry 2023-06-24 [PMID: 37390510] (Bioassay, N/A)
• AK Solanki, E Arif, P Srivastava, CM Furcht, B Rahman, P Wen, A Singh, LB Holzman, WR Fitzgibbon, MN Budisavlje, GP Lobo, SH Kwon, Z Han, MJ Lazzara, JH Lipschutz, D Nihalani Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair The Journal of Biological Chemistry, 2021-08-13;0(0):101079. 2021-08-13 [PMID: 34391780] (Bioassay, Human)
• Z Fan, Y Tian, Z Chen, L Liu, Q Zhou, J He, J Coleman, C Dong, N Li, J Huang, C Xu, Z Zhang, S Gao, P Zhou, K Ding, L Chen Blocking interaction between SHP2 and PD-1 denotes a novel opportunity for developing PD-1 inhibitors EMBO Mol Med, 2020-05-11;0(0):e11571. 2020-05-11 [PMID: 32391629] (ELISA Capture, Human)
• 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 The tyrosine phosphatase SHP2 controls TGF?-induced STAT3 signaling to regulate fibroblast activation and fibrosis Nat Commun, 2018-08-14;9(1):3259. 2018-08-14 [PMID: 30108215] (Enzyme Assay, Human)
• Zang Q, Martinez B, Yao X, Maass D, Ma L, Wolf S, Minei J Sepsis-induced cardiac mitochondrial dysfunction involves altered mitochondrial-localization of tyrosine kinase Src and tyrosine phosphatase SHP2. PLoS ONE, 2012-08-27;7(8):e43424. 2012-08-27 [PMID: 22952679] (Bioassay, Rat)

Bioinformatics

• Gene Symbol: PTPN11
• Uniprot:
  • NP_002825.3()