Recombinant Human Renin Protein, CF Summary
Product Specifications
Leu24-Arg406, 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.
4090-AS
| 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
- Activation Buffer: 5 mM Tris, 15 mM NaCl, 1 mM CaCl2, 0.005% Brij-35, pH 7.5
- Assay Buffer: 50 mM Sodium Acetate, 150 mM NaCl, 2 mM EDTA, pH 5.0
- Recombinant Human Renin (rhRenin) (Catalog # 4090-AS)
- Recombinant Human Active Trypsin 3/PRSS3 (rhTrypsin 3) (Catalog # 3714-SE)
- Substrate: Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(dabcyl)-Arg, 1 mM stock in DMSO
- 4-(2-Aminoethyl-benzensulfonyl fluoride hydrochloride) (AEBSF) (Catalog # EI001), 100 mM stock in deionized water.
- Black 96 well Plate
- Plate Reader with Fluorescence Read Capability
- Dilute rhRenin to 0.2 mg/mL in Activation Buffer.
- Dilute rhTrypsin 3 to 4 µg/mL in Activation Buffer.
- Mix equal volumes of the diluted rhRenin and Trypsin.
- Incubate at 37 °C for 1 hour to activate rhRenin.
- Stop activation with AEBSF at 1 mM and incubate at room temperature for 30 min.
- Dilute Substrate to 20 µM in Assay Buffer.
- Dilute rhRenin to 20 µg/mL in Assay Buffer.
- Load into a black well plate 50 µL of 20 µg/mL of rhRenin and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank by combining 50 µL of 20 µM Substrate with 50 µL of Assay Buffer.
- Read at excitation and emission wavelengths of 350 nm and 490 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 Fmoc-Glu(EDANS)-OH.
- rhRenin: 1.0 µg
- Substrate: 10 µM
Reconstitution Calculator
Background: Renin
Human Renin is a member of the aspartyl proteinase family produced largely in part by the juxtaglomerular cells in the kidney (1). Renin differs from the other members of this class by having a pH optimum near the neutral pH region with native substrates instead of a pH 2.0 to 3.4 range (2). This more neutral pH optimum allows it to be functional in the plasma. Renin also has a very high selectivity for substrates due to a long peptide recognition on either side of the peptide bond undergoing cleavage. An octapeptide substrate was the minimum length to be cleaved by Renin. Renin plays a crucial role in the regulation of blood pressure and salt balance through the cleavage of angiotensinogen, which is the only known physiological substrate of Renin. Renin releases the decapeptide angiotensin I, which in turn is further converted to vasoactive hormone angiotensin II by angiotensin converting enzyme (ACE). Renin is produced as prorenin with 43 pro residues at the N‑terminal of mature Renin. The inactive prorenin becomes activated proteolytically by trypsin, cathepsin B, or other proteinases.
- Yokosawa, H. et al. (1980) J. Biol. Chem. 255:3498.
- Fuminaki, S. et al. (2004) in Handbook of Proteolytic Enzymes, Barret, A. J. et al. eds. p. 54.
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