Recombinant Human Carboxypeptidase B1/CPB1 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Carboxypeptidase B1/CPB1 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a colorimetric peptide substrate, Hippuryl-Arg. The specific activity is >1,000 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Carboxypeptidase B1/CPB1 protein
  His16-Tyr417, with a C-terminal 10-His tag
• Accession #: P15086
• N-terminal Sequence: His16
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: CPB1
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 47 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 45 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in MES and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain
• 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
  1. Dilute rhCPB1 to 100 μg/mL with 1.0 μg/mL Trypsin in Activation Buffer.
  2. Incubate at room temperature for 30 minutes.
  3. Dilute active rhCPB1 to 2 μg/mL in Assay Buffer.
  4. Dilute Substrate to 2 mM in Assay Buffer.
  5. Load 50 μL of the 2 μg/mL rhCPB1 into a plate. Include a Substrate blank with 50 μL of Assay Buffer.
  6. Start the reaction by adding 50 μL of diluted Substrate into wells.
  7. Read in kinetic mode for 5 minutes at an absorbance of 254 nm.
  8. 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).

  Per Well:
  • rhCPB1: 0.1 μg
  • Substrate: 1 mM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Carboxypeptidase B1/CPB1 Protein, CF

• carboxypeptidase B
• carboxypeptidase B1 (tissue)
• Carboxypeptidase B1
• CPB
• CPB1
• DKFZp779K1333
• EC 3.4.17
• EC 3.4.17.2
• Pancreas-specific protein
• PASP
• PCPB
• procarboxypeptidase B

Background

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).

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.
2. Yamamoto, K.K. et al. (1992) J. Biol. Chem. 267:2575.

FAQs for Carboxypeptidase B1/CPB1 (2897-ZN). (Showing 1 - 2 of 2 FAQ).

1. I use porcine carboxypeptidase B (CPB) in my fermentation process. I want to detect the residual CPB in my finished product preferably by ELISA/western blot. How will antibody pairs from Novus Biologics help me do that? Is a colorimetric assay possible with the antibody pair?
   • Any detection system can be employed when using the antibody pairs. The detection system will depend on the secondary used against the detection primary antibody from the pair, and the conjugate on the secondary antibody. The pair will work effectively in capturing the protein of interest, and subsequently detecting it with the second antibody in the pair for ELISA. This pair is not evaluated in Western Blot directly, but there Novus carries similar primary antibodies that we have that are validated in that application.
2. I am trying to establish ELISA detection for carboxy peptidase. I am using a rat recombinant carboxypeptodase b from Roche diagnostics (cat no.03358682103). Can you please suggest the suitable kit .
   • Here is a link to our CPB1 products. H00001360-AP21 and H00001360-AP11 may suit your needs.

Bioinformatics

• Gene Symbol: CPB1
• Uniprot:
  • P15086()