Recombinant Human VHR Protein, CF
Recombinant Human VHR Protein, CF Summary
Product Specifications
Ser2-Pro185, with an N-terminal Met and 6-His tag
Analysis
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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.
1654-VH
| Formulation | Supplied as a 0.2 μm filtered solution in HEPES, NaCl, DTT and Glycerol. |
| 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 Procedure
- Assay Buffer: 50 mM HEPES, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mg/mL BSA, pH 7.5
- Recombinant Human VHR (rhVHR) (Catalog # 1654-VH)
- Substrate: 3-O-Methyl Fluorescein Phosphate (OMFP) (Sigma, Catalog # M2629), 2.5 mM stock in DMSO
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhVHR to 5 ng/µL in Assay Buffer.
- Dilute substrate OMFP to 500 µM in Assay Buffer. The solution will be light yellow.
- Load in a clear plate 50 µL of 5 ng/µL rhVHR, and start the reaction by adding 50 µL of 500 µM Substrate. Include a Substrate Blank containing 50 µL of the Assay Buffer and 50 µL of Substrate.
- Read at a wavelength of 477 nm (absorbance) in kinetic mode for 5 minutes.
- Calculate specific activity:
|
Specific Activity (μmol/min/mg) = |
Adjusted Vmax* (OD/min) x well volume (L) x 106 μmol/mol |
| ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (mg) |
*Adjusted for Substrate Blank
**Using the extinction coefficient 27,200 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD. Per Well:
- rhVHR: 0.00025 mg
- Substrate: 250 µM
Reconstitution Calculator
Background: VHR
Vaccinia Virus VH1-related Phosphatase (VHR), also known as Dual-Specificity Phosphatase 3 (DUSP3), removes inorganic phosphate groups covalently attached to tyrosine, serine and threonine residues in proteins (1). It belongs to a family of phosphatases that selectively dephosphorylate MAP kinases. VHR has been shown to dephosphorylate cellular ERK1, ERK2 and JNK, but not p38 (2, 3). Phosphorylation of VHR at tyrosine 138 by ZAP-70 enhances inhibition of ERK2, suggesting a role for VHR in modulating T-cell receptor responses (3). The enzymatic mechanism for VHR has been studied in detail (4) and its X-ray crystal structure has been characterized (5). Its well-defined biochemistry has made it useful in screening assays for compounds that inhibit phosphatases (6).
- Ishibashi, T. et al. (1992) Proc. Natl. Acad. Sci. USA 89:12170.
- Todd, J.L. et al. (1999) J. Biol. Chem. 274:13271.
- Alonso, A. et al. (2003) Nat. Immunol. 4:44.
- Zhang, Z.-Y. et al. (1995) Biochemistry 34:16088.
- Yuvaniyama, J. et al. (1996) Science 272:1328.
- Ueda, K. et al. (2002) FEBS Lett. 525:48.
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