Recombinant Human Meprin beta Subunit/MEP1B Protein, CF
Recombinant Human Meprin beta Subunit/MEP1B Protein, CF Summary
Product Specifications
Leu21-Ser593 & Thr23-Ser593, both 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.
2895-ZN
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. |
| Shipping | The product is shipped with dry ice or equivalent. 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 Human Meprin beta Subunit/MEP1B (rhMEP1B) (Catalog # 2895-ZN)
- Trypsin (Sigma, Catalog # T-1426 )
- AEBSF (Catalog # EI001), 100 mM stock in water
- Substrate: MCA-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Arg-Lys(DNP)-Arg-Arg-NH2 (Catalog # ES004)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhMEP1B to 100 µg/mL with 1 µg/mL Trypsin in Activation Buffer.
- Incubate for 45 minutes at 37 °C. Stop Trypsin activation by adding AEBSF at a final concentration of 1 mM in Activation Buffer and mixing well.
- Dilute activated rhMEP1B to 0.4 µg/mL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of 0.4 µg/mL rhMEP1B into plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 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).
- rhMEP1B: 0.020 µg
- Substrate: 10 µM
Reconstitution Calculator
Background: Meprin beta Subunit/MEP1B
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 heterooligomers, which are also referred to as Merpin A (composed of alpha subunits with or without beta subunits) and Merpin 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 701 amino acid sequence of human Merpin beta subunit precursor consists of a signal peptide (residues 1 to 21), a pro region (residues 22 to 61), and a mature chain (residues 62 to 701) containing the following domains, catalytic (residues 62 to 259), MAM (residues 260 to 429), MATH (residues 430 to 585), EGF-like (residues 604 to 644), transmembrane (residues 653 to 673), and cytoplasmic (residues 674 to 701). The pro enzyme terminating at residue 593 was expressed and the secreted protein purified from conditioned medium. After trypsin treatment, the activated enzyme cleaved a flurogenic peptide, which contains Asp and Glu, the preferred residues found in the P1’ and P1 sites (3).
- 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.
Citations for Recombinant Human Meprin beta Subunit/MEP1B 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.
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Citations: Showing 1 - 2
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Meprin ? contributes to collagen deposition in lung fibrosis
Authors: V Biasin, M Wygrecka, LM Marsh, C Becker-Pau, L Brcic, B Ghanim, W Klepetko, A Olschewski, G Kwapiszews
Sci Rep, 2017-01-06;7(0):39969.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay -
Astacin proteases cleave dentin sialophosphoprotein (Dspp) to generate dentin phosphoprotein (Dpp).
Authors: Tsuchiya S, Simmer JP, Hu JC, Richardson AS, Yamakoshi F, Yamakoshi Y
J. Bone Miner. Res., 2011-01-01;26(0):220.
Species: Human
Sample Types:
Applications: Bioassay
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