Recombinant Mouse Carbonic Anhydrase XIV Protein, CF Summary
Ala16-Met290, with a C-terminal 10-His tag
Accession # Q9WVT6
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, pH 7.5
- Recombinant Mouse Carbonic Anhydrase XIV/CA14 (rmCA14) (Catalog # 2504-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: Spectra-Max Plus by Molecular Devices) or equivalent
- Dilute rmCA14 to 16 ng/µL in Assay Buffer.
- Dilute Substrate to 2 mM in Assay Buffer.
- Load in a plate 50 µL of 16 ng/µL rmCA14, 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).
- rmCA14: 0.8 µg
- Substrate: 1 mM
Background: Carbonic Anhydrase XIV/CA14
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 (6th International Conference on the CAs, June 20‑25, 2003, Slovakia) ranged from use of CAs as markers for tumor and hypoxia in clinic, as nutritional supplement in milk, and as a tool for CO2 removal and mosquito control in industry. Mouse CA14 is a type I membrane enzyme highly expressed in the kidney and heart, followed by the skeletal muscle, brain, lung and liver (2). The deduced amino acid sequence of mouse CA14 consists of a signal peptide (residues 1 to 15), an ectodomain (residues 16 to 290), a transmembrane domain (residues 291 to 311) and a cytoplamic region (residues 32 to 337). rmCA14 corresponds to the ectodomain and has activity as described in Activity Assay Protocol.
- Hewett-Emmett, D. and R.E. Tashian (1996) Mol. Phylogenet. Evol. 5:50.
- Mori, K. et al. (1999) J. Biol. Chem. 274:15701.
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