Recombinant Human Chymase/CMA1 Protein, CF Summary
Met1-Asn247, with a C-terminal 10-His tag
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 HEPES and NaCl.|
|Shipping||The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.|
|Stability & Storage:||Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
- Maturation Buffer: 50 mM MES, pH 5.5
- Cathepsin Buffer: 50 mM MES, 50 mM NaCl, 5 mM DTT, pH 5.5
- Assay Buffer: 20 mM Tris, 2 M KCl, 0.02% (v/v) Triton® X-100, pH 9.0
- Recombinant Human Chymase/CMA1 (rhChymase) (Catalog # 4099-SE)
- Recombinant Mouse Active Cathepsin C/DPPI (rmCathepsin C) (Catalog # 2336-CY)
- Heparin (Sigma, Catalog # H3393), 20 mg/mL stock in deionized water
- N-Ethylmaleimide (NEM) (Sigma, Catalog # E1271), 50 mM stock in deionized water
- Substrate: SUC-Ala-Ala-Pro-Phe-AMC (Bachem, Catalog # I-1465), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhChymase to 20 µg/mL in Maturation Buffer.
- Dilute rmCathepsin C to 20 µg/mL in Cathepsin Buffer.
- Dilute Heparin to 1.5 mg/mL in deionized water.
- Combine 15 µL of 20 µg/mL rhChymase and 15 µL of 20 µg/mL rmCathepsin C.
- Add 1 µL of Heparin and mix well.
- Incubate at room temperature for 1 hour.
- Stop maturation by adding 1.98 µL of 50 mM stock of NEM for a final concentration of 3 mM.
- Dilute rhChymase to 2 µg/mL in Assay Buffer.
- Incubate at room temperature for 5 minutes.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load 50 µL of 2 µg/mL rhChymase in a plate, and start the reaction by adding 50 µL of 200 µM Substrate. As a Substrate Blank combine 50 μL of Substrate with 50 μL of Assay Buffer.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
- Calculate Specific Activity:
Specific Activity (pmol/min/µg) =
|Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)|
|amount of enzyme (µg)|
*Adjusted for Substrate Blank
**Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).
- rhChymase: 0.100 µg
- Substrate: 100 µM
Chymases are a group of chymotrypsin-like serine proteases secreted by mast cells (1). They are synthesized as inactive precursors containing a 2-residue propeptide, which needs to be removed by dipeptidyl peptidase I/cathepsin C for the enzymatic activity (2). Human Chymase encoded by the CMA1 gene is known to be involved in hypertention and heart failure through its ability to convert angiotensin I (Ang I) to angiotensin II (Ang II), which plays a key role in the regulation of arterial pressure (3). In addition, it is also important in physiological and pathological conditions including inflammation, fibrosis and processing of cytokines (4). Therefore, designing a specific inhibitor for Chymase activity has been a pharmacologic strategy to develop therapeutic agents.
- Caughey, G.H. (2004) in Handbook of Proteolytic Enzymes. Barrett, A.J. et al. ed. p. 1531, Academic Press, San Diego.
- Murakami, M. et al. (1995) J. Biol. Chem. 270:2218.
- Miyazaki, M. and S. Takai (2006) J. Pharmacol. Sci. 100:391.
- Nakajima, M. and N. Naya (2002) Jpn. J. Pharmacol. 90:206.
Product Specific NoticesTriton is a registered trademark of Union Carbide Corp.
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