Recombinant Human Carbonic Anhydrase VB Protein, CF

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

Recombinant Human Carbonic Anhydrase VB Protein, CF Summary

• Details of Functionality: Measured by its esterase activity. The specific activity is >150 pmol/min/µg, as measured under the described conditions.
• Source: E. coli-derived human Carbonic Anhydrase VB/CA5B protein
  Cys34-Pro317, with a C-terminal 10-His tag
• Accession #: NP_009151
• N-terminal Sequence: Cys34 & Ser35
• Protein/Peptide Type: Recombinant Enzymes
• Gene: CA5B
• 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: 34 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: 34 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 12.5 mM Tris, 75 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5
  • Recombinant Human Carbonic Anhydrase VB (rhCA5B) (Catalog # 3176-CA)
  • Substrate: 4-Nitrophenyl Acetate (4-NPA) (Sigma, Catalog # N8130), 100 mM stock in acetone
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rhCA5B to 2 ng/µL in Assay Buffer.
  2. Dilute substrate to 2 mM in Assay Buffer.
  3. Load in plate, 50 µL of 2 ng/µL rhCA5B, and start the reaction by adding 50 µL of 2 mM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 2 mM Substrate.
  4. Read at a wavelength of 400 nm (bottom read) in kinetic mode for 5 minutes.
  5. Calculate specific activity:

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

  *Adjusted for Substrate Blank
  **Derived using calibration standard 4-Nitrophenol (Sigma, Catalog # 241326).

  Per Well:
  • rhCA5B: 0.1 µg
  • Substrate: 1 mM

Notes

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

Alternate Names for Recombinant Human Carbonic Anhydrase VB Protein, CF

• CA5B
• Carbonate dehydratase VB
• carbonic anhydrase 5B, mitochondrial
• Carbonic Anhydrase VB
• carbonic anhydrase VB, mitochondrial
• carbonic dehydratase
• CAVB
• CA-VB
• EC 4.2.1.1
• MGC39962

Background

Carbonic Anhydrase (CA) catalyzes the reversible reaction of CO₂ + H₂O = HCO₃^- + H⁺, which is fundamental to many processes such as respiration, renal tubular acidification and bone resorption (1). Topics in a CA meeting (6^th International Conference on the CAs, June 20 - 25, 2003, Slovakia) ranged from the use of CAs as markers for tumor and hypoxia in the clinic, as a nutritional supplement in milk, and as a tool for CO₂ removal and mosquito control in industry. Carbonic Anhydrase VB encoded by the CA5B gene is a mitochondrial protein. In comparison with another mitochondrial CA (CA5A), CA5B has different tissue distribution and chromosomal location (2, 3). The amino acid sequence of human CA5B (residues 34 to 317) is 94%, 93%, 92%, and 74% identical to that of canine, mouse, bovine/rat, and chicken.

1. Hewett-Emmett, D. and R.E. Tashian (1996) Mol. Phylogenet. Evol. 5:50.
   
2. Fujikawa-Adachi, K. et al. (1999) J. Biol. Chem. 274:21228.
3. Shah, G.N. et al. (2000) Proc. Natl. Acad. Sci. USA 97:1677.

Bioinformatics

• Gene Symbol: CA5B
• Uniprot:
  • NP_009151()