Recombinant Human Carbonic Anhydrase III Protein, CF
Recombinant Human Carbonic Anhydrase III Protein, CF Summary
Product Specifications
Ala2-Lys260
Analysis
Product Datasheets
Carrier Free
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.
2185-CA
| Formulation | Lyophilized from a 0.2 μm filtered solution in Tris and NaCl. |
| Reconstitution | Reconstitute at 1 mg/mL in sterile 25 mM Tris and 150 mM NaCl, pH 7.5. |
| Shipping | The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 12.5 mM Tris, 75 mM NaCl, pH 7.5
- Recombinant Human Carbonic Anhydrase III/CA3 (rhCA3) (Catalog # 2185-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 rhCA3 to 400 ng/µL in Assay Buffer.
- Dilute Substrate to 2.0 mM in Assay Buffer.
- In a plate load 50 µL of 400 ng/µL rhCA3, and include a Substrate Blank containing 50 µL Assay Buffer.
- Start the reaction by adding 50 µL of 2.0 mM Substrate to wells containing rhCA3 and Assay Buffer.
- 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).
- rhCA3: 20 µg
- Substrate: 1.0 mM
Background: Carbonic Anhydrase III/CA3
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 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. CA3 is a cytosolic enzyme with a very low CA activity. It is expressed at low levels in human muscle during early development but increases rapidly during the last trimester to reach 50-60% of adult levels at birth (2).
- Hewett-Emmett, D. and R.E. Tashian (1996) Mol. Phylogenet. Evol. 5:50.
- Sly, W.S. and P.Y. Hu (1995) Annu. Rev. Biochem. 64:375.
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