Recombinant Mouse Meprin alpha Subunit/MEP1A Protein, CF
Recombinant Mouse Meprin alpha Subunit/MEP1A Protein, CF Summary
Product Specifications
Val34-Arg615, 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.
4007-ZN
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. |
| 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, 1 M NaCl, pH 9.0
- Recombinant Mouse Meprin alpha Subunit/MEP1A (rmMEP1A) (Catalog # 4007-ZN)
- Trypsin (Sigma, Catalog # T-1426)
- AEBSF (Catalog # EI001), 100 mM stock in deionized water
- Substrate: MCA-Tyr-Val-Ala-Asp-Ala-Pro-Lys(DNP)-OH (Catalog # ES007)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rmMEP1A to 100 µg/mL in Activation Buffer with Trypsin at 0.1 µg/mL.
- Incubate for 45 minutes at 37 °C.
- Stop Trypsin activation by adding an equal volume of AEBSF at 2 mM in Activation Buffer and mixing well.
Dilute Substrate to 40 µM in Assay Buffer. - Dilute activated rmMEP1A to 0.8 µg/mL in Assay Buffer.
- Load in a plate 50 µL of 0.8 µg/mL rmMEP1A, and start the reaction by adding 50 µL of 40 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 40 µM Substrate.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU) |
| amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).
- rmMEP1A: 0.04 µg
- Substrate: 20 µM
Reconstitution Calculator
Background: Meprin alpha Subunit/MEP1A
Meprins are multimeric proteases composed of alpha and beta subunits, which are members of the astacin family of zinc endopeptidases (1, 2). Both subunits form disulfide‑linked homo‑ or hetero‑oligomers, which are also referred to as Meprin A (composed of alpha subunits with or without beta subunits) and Meprin B (composed of beta subunits only) (3). Although the two subunits share 42% identity in their amino acid sequence, they differ significantly in their oligomeric structure, post‑translational processing and subsequently cellular location, and substrate and peptide bond specificity (4). The 760 amino acid sequence of mouse meprin alpha subunit precursor consists of a signal peptide (residues 1‑33), a pro region (residues 34‑77), and a mature chain (residues 78‑760) containing the following domains, catalytic (residues 78‑275), MAM (residues 276‑445), MATH (residues 447‑607), EGF‑like (residues 684‑724), transmembrane (residues 727‑754), and cytoplasmic (residues 755‑760) (5). The pro enzyme terminating at residue 615 was expressed and the secreted protein purified from conditioned medium. The molecular masses of recombinant mouse MEP1A are similar to those observed for the alpha subunit of rat Meprin A (6).
- Bond, J.S. and R.J. Beynon (1995) Protein Sci. 4:1247.
- Stocker, W. et al. (1995) Protein Sci. 4:823.
- Bertenshaw, G.P. et al. (2001) J. Biol. Chem. 276:13248.
- Ishmael, F.T. et al. (2005) J. Biol. Chem. 280:13895.
- Jiang, W. et al. (1992) J. Biol. Chem. 267:9185.
- Bertenshaw, G.P. et al. (2003) J. Biol. Chem. 278:2522.
Citation for Recombinant Mouse Meprin alpha Subunit/MEP1A 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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Successive action of meprin A and neprilysin catabolizes B-type natriuretic peptide.
Authors: Pankow K, Wang Y, Gembardt F, Krause E, Sun X, Krause G, Schultheiss HP, Siems WE, Walther T
Circ. Res., 2007-09-06;101(9):875-82.
Applications: Bioassay
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