Recombinant Human TC-PTP (aa 2-314) Protein, CF Summary
Thr2-Asn314, with an N-terminal Met and 6-His tag
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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 HEPES, NaCl, Glycerol and Betamercaptoethanol.|
|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: 10 mM HEPES, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mg/mL BSA, 1 mM dithothreitol, pH 7.5
- Recombinant Human TC‑PTP (rhTC-PTP) (Catalog # 1930-PT)
- 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 # 92592) or equivalent
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Phosphate Standard: 1 M KH2PO4
- Dilute rhTC-PTP to 0.025 µg/mL in Assay Buffer.
- Load 40 µL of diluted rhTC-PTP to a plate. Include Substrate Blanks containing 40 µL Assay Buffer in place of rhTC-PTP.
- Dilute the Substrate in Assay Buffer to 1 mM and add 10 µL to the wells.
- Cover the plate with parafilm or a plate sealer and incubate at 30 °C for 30 minutes.
- Prepare a standard curve from the 1M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of assay buffer for a 10 mM stock. Continue by adding 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.) Perform six additional one-half serial dilutions of the 100 µM Phosphate stock. The standard curve has a range of 0.078 to 5 nmol per well.
- Load 50 µL of the standards and blanks to the plate.
- Add 10 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
- Add 10 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- 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:
- rhTC-PTP: 0.000001 mg (1 ng)
- Substrate: 200 µM
T-cell protein tyrosine phosphatase (TC-PTP), also known as PTPT and PTPN2, is an enzyme that removes phosphate groups covalently attached to tyrosine residues in proteins. This enzyme has two C-terminal end splice variants with distinctly different subcellular localizations. The shorter 45 kilodalton isoform is exclusively nuclear in resting cells, but redistrubutes to the cytosol upon stimulation with growth factors (1) and cellular stress (2). The longer 48 kilodalton isoform is exclusively found in the endoplasmic reticulum (3) and seems to have distinctly different physiologic substrates from the smaller isoform (1, 4). Although found in many cell types and tissues, TC-PTP is particularly prominent in hemopoietic cell types (5, 6). Knockout mice lacking TC-PTP are born viable but die 3 to 5 weeks after birth of erythropoietic and lymphopoietic deficits (7), indicating a critical role for TC-PTP in bone marrow maturation. TC-PTP will dephosphorylate a wide range of phosphoproteins, such as p52Shc (6) and receptors for EGF (1), Insulin (8) and growth hormone (6). The recombinant protein lacks the C-terminal 100 amino acids that determine intracellular localization but is fully active (9).
- Tiganis, T. et al. (1999) J. Biol. Chem. 274:27768.
- Lam, M.H. et al. (2001) J. Biol. Chem. 276:37700.
- Lorenzen, J.A. et al. (1995) J. Cell Biol. 131:631.
- Tiganis, T. et al. (1998) Mol. Cell. Biol. 18:1622.
- Cool, D.E. et al. (1989) Proc. Natl. Acad. Sci. USA 86:5257.
- Pasquali, C. et al. (2003) Mol. Endocrinol. 17:2228.
- You-Ten, K.E. et al. (1997) J. Exp. Med. 186:683.
- Galic, S. et al. (2003) Mol. Cell. Biol. 23:2096.
- Cool, D.E. et al. (1990) Proc. Natl. Acad. Sci. USA 87:7280.
Citations for Recombinant Human TC-PTP (aa 2-314) 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 - 3
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Benzoquinone, a leukemogenic metabolite of benzene, catalytically inhibits the protein tyrosine phosphatase PTPN2 and alters STAT1 signaling
Authors: R Duval, LC Bui, C Mathieu, Q Nian, J Berthelet, X Xu, I Haddad, J Vinh, JM Dupret, F Busi, F Guidez, C Chomienne, F Rodrigues-
J. Biol. Chem., 2019;0(0):.
Applications: Western Blot
Field sampling marine plankton for biodiscovery
Authors: RA Ingebrigts, E Hansen, JH Andersen, HC Eilertsen
Sci Rep, 2017;7(1):15863.
Sample Types: Whole Cells
Integrin alpha1beta1 promotes caveolin-1 dephosphorylation by activating T cell protein-tyrosine phosphatase.
Authors: Borza CM, Chen X, Mathew S, Mont S, Sanders CR, Zent R, Pozzi A
J. Biol. Chem., 2010;285(51):40114-24.
Sample Types: Whole Cells
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Phosphatase Activity Assays
Phosphatase Peptide Substrates
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