Recombinant Human Carboxypeptidase B1/CPB1 Protein, CF
Recombinant Human Carboxypeptidase B1/CPB1 Protein, CF Summary
Product Specifications
His16-Tyr417, 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.
2897-ZN
| Formulation | Supplied as a 0.2 μm filtered solution in MES 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
- Activation Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl2, 0.05% Brij-35, pH 7.5 (TCNB)
- Assay Buffer: 50 mM Tris, 100 mM NaCl, pH 7.5
- Recombinant Human Carboxypeptidase B1/CPB1 (rhCPB1) (Catalog # 2897-ZN)
- Trypsin (Sigma, Catalog # T-1426)
- Substrate: Hippuryl-R (Sigma, Catalog # H-2508), 25 mM in diH2O
- 96 well clear UV-transparent microplate (Corning, Catalog # 3635)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhCPB1 to 100 μg/mL with 1.0 μg/mL Trypsin in Activation Buffer.
- Incubate at room temperature for 30 minutes.
- Dilute active rhCPB1 to 2 μg/mL in Assay Buffer.
- Dilute Substrate to 2 mM in Assay Buffer.
- Load 50 μL of the 2 μg/mL rhCPB1 into a plate. Include a Substrate blank with 50 μL of Assay Buffer.
- Start the reaction by adding 50 μL of diluted Substrate into wells.
- Read in kinetic mode for 5 minutes at an absorbance of 254 nm.
- Calculate specific activity using the following formula:
|
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 Hippuric acid (Sigma, Catalog # 112003).
- rhCPB1: 0.1 μg
- Substrate: 1 mM
Reconstitution Calculator
Background: Carboxypeptidase B1/CPB1
Carboxypeptidase B1, encoded by the CPB1 gene, specifically cleaves the C-terminal Arg and Lys residues with a preference for Arg (1). Expressed mainly in pancreas, CPB1 is a useful serum marker for acute pancreatitis (2). The deduced amino acid sequence of human CPB1 consists of a signal peptide (residues 1 to 15), a pro region (residue 16 to 110), and a mature chain (residues 111 to 417). The purified rhCPB1 corresponds to the pro form, which can be activated by trypsin, the only pancreatic protease capable of generating active enzyme from the zymogen in vitro (1).
- Aviles, F.X. and J. Vendrell (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, A.J. et al.) p. 831, Academic Press, San Diego.
- Yamamoto, K.K. et al. (1992) J. Biol. Chem. 267:2575.
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