Recombinant Human PTP1B Protein, CF Summary
Product Specifications
Glu2-Asn321, with an N-terminal Met and 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
1366-PT
| Formulation | Lyophilized from a 0.2 μm filtered solution in HEPES, NaCl and DTT. |
| Reconstitution | 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. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
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
- Dilute rhPTP1B to 0.0625 μg/mL in Assay Buffer.
- Load 40 µL of diluted rhPTP1B to a 96 well clear plate with 40 µL Assay Buffer as blanks.
- Dilute the Substrate to 500 µM 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 1 M Phosphate Standard supplied in the Malachite Green Phosphate Detection Kit.
- Add 10 μL of the 1 M Phosphate Standard to 990 μL of Assay Buffer for a 10 mM stock.
- 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).
- 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.
- After the 30 °C incubation, transfer (in duplicate) 50 μL of the standards and blanks (Assay Buffer) to the plate.
- Add 10 μL of the Malachite Green Reagent A to each sample, standard, and blank. Mix and incubate for 10 minutes at room temperature.
- Add 10 μL of the Malachite Green Reagent B to each sample, standard, and blank. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) 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 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
Reconstitution Calculator
Background: PTP1B
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.
- Angers-Loustau, et al. (1999) Biochem. Cell Biol. 77:493.
- Sarmiento, et al. (1998) J. Biol. Chem. 273:26368.
- Bjorge, et al. (2000) J. Biol. Chem. 52:41439.
- Balsamo, et al. (1996) J. Cell Biol. 134:801.
- Elchebly, et al. (1999) Science 283:1544.
Citations for Recombinant Human PTP1B 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.
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Citations: Showing 1 - 2
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Identification of a small molecular insulin receptor agonist with potent antidiabetes activity.
Authors: 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
Diabetes, 2014-04-01;63(4):1394-409.
Species: Human
Sample Types: Protein
Applications: Bioassay -
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., 2010-10-12;285(51):40114-24.
Species: Mouse
Sample Types: N/A, Whole Cells
Applications: Bioassay, Western Blot
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