Recombinant Human Carboxypeptidase M Protein, CF
Recombinant Human Carboxypeptidase M Protein, CF Summary
Product Specifications
Leu18-Ser423, with a C-terminal 6-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.
7457-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
- Assay Buffer: 50 mM MES, 0.2% Triton® X-100, 5 mM CaCl2, pH 6.0
- Recombinant Human Carboxypeptidase M (rhCPM) (Catalog # 7457-ZN)
- Substrate: Bz-Ala-Arg-OH (Bachem, Catalog # G-4145), 50 mM stock in DMSO
- 2-Mercaptoethanol (Sigma, Catalog # M7154)
- NaOH, 2 M stock in deionized water
- o-Phthaldialdehyde (OPA) (Sigma, Catalog # P0657), 0.373 M in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhCPM to 0.04 µg/mL in Assay Buffer.
- Dilute Substrate to 1 mM in Assay Buffer.
- Mix (in duplicate) 150 μL of 0.04 µg/mL rhCPM and 150 μL 1 mM Substrate for a final concentration of 0.02 µg/mL and 500 µM respectively. Include controls containing 150 μL of 1 mM Substrate only.
Incubate for 10 minutes at room temperature. - Stop reaction by adding 300 μL of a solution containing 15 mM o-PA in 0.2 M NaOH containing 0.1% (v/v) 2‑Mercaptoethanol and mix well.
- Add 150 μL of 0.04 µg/mL rhCPM to controls after stopping the reaction.
- Incubate all for 10 minutes at room temperature.
- Load 200 µL of the incubated samples in triplicate into the plate.
- Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
- Calculate Specific Activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU) |
| Incubation time (min) x amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard L-Arginine (Sigma, Catalog # A5006).
- rhCPM: 0.002 µg
- Substrate: 0.25 mM
- o-PA: 7.5 mM
Reconstitution Calculator
Background: Carboxypeptidase M
Carboxypeptidase M (CPM) is a zinc metallopeptidase specific for the removal of arginine and lysine residues from peptides. CPM is bound to the plasma membrane through a glycosylphosphatidylinositol anchor (1). The enzyme is thought to regulate the actions of some peptide hormones at the cell surface through their degradation (2). CPM is a biomarker for well-differentiated liposarcomas (3) and is also a marker for the maturation of monocytes to macrophages (4). CPM binds to the kinin B1 receptor on the cell surface, forming a complex that potentiates the signaling ability of the kinin B1 receptor (5). Recombinant human CPM was expressed as a C-terminally truncated protein to prevent the formation of the glycosylphosphatidylinositol anchor, resulting in the secretion of the protein.
- Tan F. et al. (2003) Biochem. J. 370:567.
- Reverter D. et al. (2004) J. Mol. Biol. 338:257.
- Erickson-Johnson M.R. et al. (2009) Mod. Pathol. 22:1541.
- Rehli M. et al. (2000) Adv. Exp. Med. Biol. 477:205.
- Zhang X. et al. (2011) J. Biol. Chem. 286:18547.
Product Specific Notices
Coomassie is a registered trademark of Imperial Chemical Industries Ltd. Triton is a registered trademark of Union Carbide Corp.Citation for Recombinant Human Carboxypeptidase M Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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Peptidylarginine Deiminase of Porphyromonas gingivalis Modulates the Interactions between Candida albicans Biofilm and Human Plasminogen and High-Molecular-Mass Kininogen
Authors: J Karkowska-, M Surowiec, M Gogol, J Koziel, B Potempa, J Potempa, A Kozik, M Rapala-Koz
Int J Mol Sci, 2020-04-03;21(7):.
Species: Human
Sample Types:
Applications: Enzyme Assay
FAQs
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