Recombinant Human VHR Protein, CF

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

Recombinant Human VHR Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a substrate, p-Nitrophenyl phosphate (pNPP). The specific activity is >100 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human VHR protein
  Ser2-Pro185, with an N-terminal Met and 6-His tag
• Accession #: Q5U0J1
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Proteins
• Gene: DUSP3
• 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: 21 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: 23 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in HEPES, NaCl, DTT and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• 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
  1. Dilute rhVHR to 20 µg/mL in Assay Buffer.
  2. Dilute Substrate to 6 mM in Assay Buffer.
  3. 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.
  4. Incubate Reactions and Enzyme Control at 37 °C for 4 hours.
  5. Load 100 µL of Reactions into a plate and stop the reactions by adding 100 µL 0.2 M NaOH.
  6. Load 200 µL of Enzyme Controls into plate.
  7. Read plate at 410 nm (absorbance) in endpoint mode.
  8. Calculate specific activity:
  
  

  Specific Activity (pmol/min/µg) = (Adjusted Abs* (OD) x Conversion Factor** (pmol/OD)) / (Incubation time (min) x amount of enzyme (µg))

  *Adjusted for Enzyme Controls
  **Derived using calibration standard p-Nitrophenol
  Per Well:
  • rhVHR: 1 µg
  • pNPP: 1.5 mM

Notes

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

Alternate Names for Recombinant Human VHR Protein, CF

• dual specificity phosphatase 3
• dual specificity protein phosphatase 3
• Dual specificity protein phosphatase VHR
• DUSP3
• EC 3.1.3.16
• EC 3.1.3.48
• Vaccinia H1-related phosphatase
• vaccinia virus phosphatase VH1-related
• VHR
• VHRserine/threonine specific protein phosphatase

Background

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).

1. Ishibashi, T. et al. (1992) Proc. Natl. Acad. Sci. USA 89:12170.
2. Todd, J.L. et al. (1999) J. Biol. Chem. 274:13271.
3. Alonso, A. et al. (2003) Nat. Immunol. 4:44.
4. Zhang, Z.-Y. et al. (1995) Biochemistry 34:16088.
5. Yuvaniyama, J. et al. (1996) Science 272:1328.
6. Ueda, K. et al. (2002) FEBS Lett. 525:48.

Bioinformatics

• Gene Symbol: DUSP3
• Uniprot:
  • Q5U0J1()