Recombinant Human PTP1B Protein, CF

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

Recombinant Human PTP1B 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 >15 μmol/min/mg, as measured under the described conditions.
• Source: E. coli-derived human PTP1B protein
  Glu2-Asn321, with an N-terminal Met and 6-His tag
• Accession #: P18031
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: PTPN1
• 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: 38 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: 38 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in HEPES, NaCl and DTT.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Reconstitution Instructions: Reconstitute to 100 μg/mL in sterile 10 mM HEPES, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM dithiothreitol, and 500 μg/mL bovine serum albumin, pH 7.5.
• Assay Procedure:
  • Assay Buffer: 10 mM HEPES, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mg/mL BSA, 1 mM dithiothreitol, pH 7.5
  • Recombinant Human PTP1B (rhPTP1B) (Catalog # 1366-PT)
  • Substrate: Asp-Ala-Asp-Glu-Tyr(PO₃)-Leu-Ile-Pro-Gln-Gln-Gly (Catalog # ES006), 10 mM stock in diH₂O
  • Malachite Green Phosphate Detection Kit (Catalog # DY996)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhPTP1B to 0.0625 μg/mL in Assay Buffer.
  2. Load 40 µL of diluted rhPTP1B to a 96 well clear plate with 40 µL Assay Buffer as blanks.
  3. Dilute the Substrate to 500 µM and add 10 µL to the wells.
  4. Cover the plate with parafilm or a plate sealer and incubate at 30 °C for 30 minutes.
  5. Prepare a standard curve from the 1 M Phosphate Standard supplied in the Malachite Green Phosphate Detection Kit.
  1. Add 10 μL of the 1 M Phosphate Standard to 990 μL of Assay Buffer for a 10 mM stock.
  2. Add 10 μL of the 10 mM phosphate stock to 990 μL of Assay Buffer for a 100 μM stock. (This is the first dilution to use as a standard).
  3. Perform six additional 1:2 serial dilutions of the 100 μM phosphate stock. The standard curve has a range of 0.078 to

     5.0 nmol per well.

  6. After the 30 °C incubation, transfer (in duplicate) 50 μL of the standards and blanks (Assay Buffer) to the plate.
  7. Add 10 μL of the Malachite Green Reagent A to each sample, standard, and blank. Mix and incubate for 10 minutes at room temperature.
  8. Add 10 μL of the Malachite Green Reagent B to each sample, standard, and blank. Mix and incubate for 20 minutes at room temperature.
  9. Read plate at 620 nm (absorbance) in endpoint mode.
  10. 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 Substrate Blank.

  Per Well:
  • Phosphate standard curve: 5.0, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, and 0 nmoles
  • rhPTP1B: 0.0000025 mg
  • Substrate: 71.4 μM

Notes

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

Alternate Names for Recombinant Human PTP1B Protein, CF

• EC 3.1.3.48
• protein tyrosine phosphatase 1B
• protein tyrosine phosphatase, non-receptor type 1
• Protein-tyrosine phosphatase 1B
• PTP1B
• PTP-1B
• PTP1Bprotein tyrosine phosphatase, placental
• PTPN1
• tyrosine-protein phosphatase non-receptor type 1

Background

Protein tyrosine phosphatase 1B (PTP1B) is an enzyme that removes phosphate groups covalently attached to tyrosine residues in proteins. This ubiquitously expressed enzyme is anchored in the endoplasmic reticulum by its C-terminal end and has its catalytic regions exposed to the cytosol. The recombinant protein lacks the C-terminal 114 amino acids but is fully active. PTP1B will dephosphorylate a wide variety of phosphoproteins, such as receptors for the growth factors insulin and epidermal growth factor (EGF), c-Src and beta -catenin. Of particular interest is the observation that PTP1B knock-out mice are resistant to high-caloric intake-induced obesity and have exaggerated insulin responses, suggesting that PTP1B may play an important role in regulating growth factor responsiveness.

1. Angers-Loustau, et al. (1999) Biochem. Cell Biol. 77:493.
2. Sarmiento, et al. (1998) J. Biol. Chem. 273:26368.
3. Bjorge, et al. (2000) J. Biol. Chem. 52:41439.
4. Balsamo, et al. (1996) J. Cell Biol. 134:801.
5. Elchebly, et al. (1999) Science 283:1544.

Publications for PTP1B/PTPN1 (1366-PT)(2)

Publications using 1366-PT

• Qiang , Guifen, Xue , Shenghui, Yang , Jenny J, Du , Guanhua, Pang , Xiaobin, Li , Xiaoting, Goswami , Devrishi, Griffin , Patrick, Ortlund , Eric A, Chan , Chi Bun, Ye , Keqiang Identification of a small molecular insulin receptor agonist with potent antidiabetes activity. Diabetes, 2014-04-01;63(4):1394-409. 2014-04-01 [PMID: 24651808] (Bioassay, Human)
• Borza CM, Chen X, Mathew S, Mont S, Sanders CR, Zent R, Pozzi A Integrin alpha1beta1 promotes caveolin-1 dephosphorylation by activating T cell protein-tyrosine phosphatase. J. Biol. Chem., 2010-10-12;285(51):40114-24. 2010-10-12 [PMID: 20940300] (Bioassay, Western Blot, Mouse)

Review for PTP1B/PTPN1 (1366-PT) (1)

Average Rating: 3

(Based on 1 review)

reviewed by: Sivanesan Dakshanamurthy SPR assay 01/30/2019

Summary

• Application: Other
• Lot: 13289001
• Comments: delayed delivery

Bioinformatics

• Gene Symbol: PTPN1
• Uniprot:
  • P18031()