Recombinant Human Renin Protein, CF

Catalog #: 4090-AS Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
4090-AS-020

R&D Systems Recombinant Proteins and Enzymes

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Recombinant Human Renin Protein, CF Summary

Product Specifications

Purity

>90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to cleave a fluorogenic peptide substrate, Arg-Glu(EDANS)-Ile-His-Pro-Phe-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-Lys(dabcyl)-Arg. The specific activity is >20 pmol/min/µg, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived human Renin protein
Leu24-Arg406, with a C-terminal 10-His tag

Accession #

P00797

N-terminal Sequence
Analysis

Leu24

Structure / Form

Pro form

Predicted Molecular Mass

44 kDa

SDS-PAGE

44 kDa and 51 kDa, reducing conditions

Product Datasheets

4090-AS

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

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 V_max* (RFU/min) x Conversion Factor** (pmol/RFU)
	amount of enzyme (µg)

*Adjusted for Substrate Blank
**Derived using calibration standard Fmoc-Glu(EDANS)-OH.

Per Well:

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.

References

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.

Entrez Gene IDs

5972 (Human); 19701 (Mouse)

Alternate Names

angiotensin-forming enzyme; Angiotensinogenase; EC 3.4.23; EC 3.4.23.15; FLJ10761; HNFJ2; Prorenin; REN; renin precursor, renal; Renin