Recombinant Mouse Carbonic Anhydrase IV Protein, CF Summary
Glu18-Ser277, with a C-terminal 10-His tag
Accession # Q64444
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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, pH 7.5
- Recombinant Mouse Carbonic Anhydrase IV/CA4 (rmCA4) (Catalog # 2414-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 rmCA4 to 80 ng/µL in Assay Buffer.
- Dilute Substrate to 2 mM in Assay Buffer.
- Load 50 µL of 80 ng/µL rmCA4 into a 96 well clear well plate, and start the reaction by adding 50 µL of 2 mM Substrate. Include a Substrate Blank containing Assay Buffer in place of rmCA4.
- 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).
- rmCA4: 4 µg
- Substrate: 1 mM
Background: Carbonic Anhydrase IV/CA4
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. The deduced amino acid sequence of mouse CA4 consists of a signal peptide (residues 1-17), an ectodomain (residues 18-277) and a pro peptide (residues 278-305), which is removed in the mature form (2). The native enzyme is attached to the membrane by a GPI-anchor. Recombinant mouse CA4 corresponds to the ectodomain and exhibits the activity as described in Activity Assay Protocol.
- Hewett-Emmett, D. and R.E. Tashian (1996) Mol. Phylogenet. Evol. 5:50.
- Tamai, S. et al. (1996) Biochem. Genet. 34:31.
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