Recombinant Mouse Carboxypeptidase A4/CPA4 Protein, CF
Recombinant Mouse Carboxypeptidase A4/CPA4 Protein, CF Summary
Product Specifications
Gly17-Tyr420, with a C-terminal 10-His tag
Accession # Q6P8K8
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.
2899-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
- Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Assay Buffer: 50 mM Tris, pH 8.0
- Recombinant Mouse Carboxypeptidase A4/CPA4 (rmCPA4) (Catalog # 2899-ZN)
- Trypsin (Sigma, Catalog # T-1426)
- AEBSF (Catalog # EI001), 100 mM in deionized water
- 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 (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rmCPA4 to 100 µg/mL with 1.0 µg/mL Trypsin in Activation Buffer.
- Incubate at room temperature for 1 hour.
- Stop Trypsin activity by adding AEBSF at a final concentration of 1 mM.
- Incubate at room temperature for 15 minutes.
- Dilute active rmCPA4 to 6 µg/mL in Assay Buffer.
- Dilute Substrate to 200 µM with 200 µM DTNB in Assay Buffer.
- Load 50 µL of the 6 µg/mL rmCPA4 into a plate, and start the reaction by adding 50 µL of Substrate/DTNB mixture. Include a Substrate Blank with 50 µL of Assay Buffer and 50 µL of Substrate/DTNB mixture.
- 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:
- rmCPA4: 0.3 µg
- Substrate: 100 µM
- DTNB: 100 µM
Reconstitution Calculator
Background: Carboxypeptidase A4/CPA4
Carboxypeptidase A4 encoded by the CPA4 gene is a member of the metallocarobxypeptidase family that cleaves the C-terminal amide or ester bond of peptides that have a free C-terminal carboxyl group. The deduced amino acid sequence of mouse CPA4 consists of a signal peptide (residues 1 to 16), a pro region (residue 17 to 113), and a mature chain (residues 114 to 420). The purified recombinant mouse CPA4 corresponds mainly to the pro form, which can be activated and assayed under the conditions described in the Activity Assay Protocol.
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