Recombinant Human Carboxypeptidase A1/CPA1 Protein, CF
Recombinant Human Carboxypeptidase A1/CPA1 Protein, CF Summary
Product Specifications
Lys17-Tyr419, 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.
2856-ZN
| 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: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij-35, pH 7.5 (TCNB)
- Recombinant Human Carboxypeptidase A1/CPA1 (rhCPA1) (Catalog # 2856-ZN)
- Trypsin (Sigma, Catalog # T-1426)
- Substrate: Ac-Phe-Thiaphe-OH (Peptides International, Catalog # STP-3621-PI),10 mM stock in DMSO
- 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB), (Sigma, Catalog # D-8130) 10 mM stock in DMSO
- 96 well clear plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhCPA1 to 100 µg/mL with 1.0 µg/mL Trypsin in Assay Buffer.
- Incubate at room temperature for 60 minutes.
Dilute active rhCPA1 to 0.2 µg/mL in Assay Buffer. - Combine equal volumes of 10 mM Substrate and 10 mM DTNB. Then, dilute this mixture to 200 µM Substrate, 200 µM DTNB with Assay Buffer.
- Load 50 µL of the 0.2 µg/mL rhCPA1 into a clear microplate. Include a substrate blank with 50 µL of Assay Buffer in place of rhCPA1.
- Start the reaction by adding 50 µL of 200 µM Substrate into wells.
- Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
- Calculate specific activity using the following formula:
|
Specific Activity (pmol/min/µg) = |
Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol |
| ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Using the extinction coefficient 13260 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD Per Well:
- rhCPA1: 0.010 µg
- Substrate: 100 µM
- DTNB: 100 µM
Reconstitution Calculator
Background: Carboxypeptidase A1/CPA1
Carboxypeptidase A1 encoded by the CPA1 gene cleaves the C-terminal amide or ester bond of peptides that have a free C-terminal carboxyl group (1). It prefers the C-terminal residues with aromatic or branched aliphatic side chains including Phe, Tyr, Trp, Leu or Ile. It is important in the degradation of food proteins to produce essential amino acids such as Phe and Trp. The deduced amino acid sequence of human CPA1 consists of a signal peptide (residues 1 to 16), a pro region (residue 17 to 110), and a mature chain (residues 111 to 419). The purified recombinant human CPA1 corresponds to the pro form, which can be activated as described in Activity Assay Protocol.
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