Recombinant Mouse Carbonic Anhydrase IX Protein, CF
Recombinant Mouse Carbonic Anhydrase IX Protein, CF Summary
Product Specifications
Gln32-Asp390, with a C-terminal 10-His tag
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.
2344-CA
| 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 Procedure
- Assay Buffer: 25 mM Tris, 150 mM NaCl, pH 7.5
- Recombinant Mouse Carbonic Anhydrase IX/CA9 (rmCA9) (Catalog # 2344-CA)
- Substrate: 4-Nitrophenyl acetate (4-NPA) (Sigma, Catalog # N8130), 100 mM stock in Actone
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rmCA9 to 4.0 ng/µL in Assay Buffer.
- Dilute Substrate to 2 mM in Assay Buffer.
- In a plate load 50 µL of 4.0 ng/µL rmCA9, and start the reaction by adding 50 µL of 2 mM Substrate to wells. Include a Substrate Blank containing 50 µL 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).
- rmCA9: 0.2 µg
- Substrate: 1 mM
Reconstitution Calculator
Background: Carbonic Anhydrase IX/CA9
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. CA9, also known as membrane antigen MN and renal cell carcinoma (RCC)-associated antigen G250, is a transmembrane enzyme expressed primarily in carcinoma cells. It is one of the best markers for hypoxia and for RCC (2, 3). Recombinant mouse CA9 corresponds to the extracellular portion of mouse CA9.
- Hewett-Emmett, D. and R.E. Tashian (1996) Mol. Phylogenet. Evol. 5:50.
- Kaluzova, M. et al. (2004) Mol. Cell Biol. 24:5757.
- Mukouyama, H. et al. (2004) Clin. Cancer Res. 10:1421.
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