Recombinant Human VHR Protein, CF Summary
Product Specifications
Ser2-Pro185, 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.
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, 10 mM DTT, pH 7.5
- Recombinant Human VHR (rhVHR) (Catalog # 1654-VH)
- Substrate: p-Nitrophenyl phosphate (pNPP), 10 mM stock in deionized water
- Sodium Hydroxide (NaOH), 0.2 M stock in deionized water
- Clear 96-well Plate (Catalog # DY990)
- Plate Reader with absorbance read capability
- Dilute rhVHR to 20 µg/mL in Assay Buffer.
- Dilute Substrate to 6 mM in Assay Buffer.
- Prepare reaction mixtures by combining equal volumes of dilute rhVHR and dilute Substrate in microtubes. Include an Enzyme Control by combining dilute rhVHR with twice the volume of 0.2 M NaOH, mix briefly; then add a volume of dilute Substrate equal the volume of dilute rhVHR. The Enzyme Control will have 2x the volume of the reaction mixture.
- Incubate Reactions and Enzyme Control at 37 °C for 4 hours.
- Load 100 µL of Reactions into a plate and stop the reactions by adding 100 µL 0.2 M NaOH.
- Load 200 µL of Enzyme Controls into plate.
- Read plate at 410 nm (absorbance) in endpoint mode.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Abs* (OD) x Conversion Factor** (pmol/OD) |
| Incubation time (min) x amount of enzyme (µg) |
**Derived using calibration standard p-Nitrophenol
- rhVHR: 1 µg
- pNPP: 1.5 mM
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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