Recombinant Human Carbonic Anhydrase VA Protein, CF Summary
Ala40-Ser305, with a C-terminal 10-His tag
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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.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris and NaCl.|
|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 Buffer: 12.5 mM Tris, 75 mM NaCl, 0.05% (v/v) Brij-35, pH 7.5
- Recombinant Human Carbonic Anhydrase VA (rhCA5A) (Catalog # 3049-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
- Dilute rhCA5A to 20 ng/µL in Assay Buffer.
- Dilute Substrate to 2 mM in Assay Buffer.
- In a plate, load 50 µL of 20 ng/µL rhCA5A, 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.
- Read at a wavelength of 400 nm (bottom read) in kinetic mode for 5 minutes.
- 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).
- rhCA5A: 1 µg
- Substrate: 1 mM
Background: Carbonic Anhydrase VA/CA5A
Carbonic Anhydrase 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 (6th 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 VA encoded by the CA5A gene is a mitochondrial protein (2, 3). In comparison with another mitochondrial CA (CA5B), CA5A has different tissue distribution and chromosomal location (4, 5). Expression and inhibitor studies of different CAs in the rat pancreatic beta cells indicate that CA5A may be involved in the regulation of insulin secretion (6). CA5A may also participate in the detoxification of ammonia produced in the gastrointestinal tract by providing bicarbonate to carbamyl phosphate synthetase I (7). The amino acid sequence of recombinant human CA5A (residues 40 to 305) is 79%, 77%, and 76% identical to that of canine, bovine, and rat/mouse.
- Hewett-Emmett, D. and R.E. Tashian (1996) Mol. Phylogenet. Evol. 5:50.
- Nagao, Y. et al. (1993) Proc. Natl. Acad. Sci. USA 90:7623.
- Nagao, Y. et al. (1995) Genomics 28:477.
- Shah, G.N. et al. (2000) Proc. Natl. Acad. Sci. USA 97:1677.
- Fujikawa-Adachi, K. et al. (1999) J. Biol. Chem. 274:21228.
- Parkkila, A.K. et al. (1998) J. Biol. Chem. 273:24620.
- Saarnio, J. et al. (1999) J. Histochem. Cytochem. 47:517.
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