Recombinant Mouse Plasma Kallikrein/KLKB1 Protein, CF

Catalog #: 2498-SE
1 Citation Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
2498-SE-010

R&D Systems Recombinant Proteins and Enzymes

1 Image

All Plasma Kallikrein/KLKB1 Proteins and Enzymes

Product Details

Citations (1)

Supplemental Products

Reviews

Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Mouse Plasma Kallikrein/KLKB1 Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Endotoxin Level

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

Activity

Measured by its ability to cleave the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The specific activity is >300 pmol/min/µg, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived mouse Plasma Kallikrein/KLKB1 protein
Gly20-Ala638, with a C-terminal 6-His tag

Accession #

NP_032481

N-terminal Sequence
Analysis

Gly20

Structure / Form

Pro form

Predicted Molecular Mass

70 kDa

SDS-PAGE

80 kDa, reducing conditions

Product Datasheets

2498-SE

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.

2498-SE

Formulation	Supplied as a 0.2 μm filtered solution in MES and NaCl.
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. 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: 50 mM Tris, 10 mM CaCl₂, 150 mM NaCl, pH 7.5 (TCN)
Assay Buffer: 50 mM Tris, 250 mM NaCl, 1 mM EDTA, pH 7.5
Recombinant Mouse Plasma Kallikrein/KLKB1 (rmKLKB1) (Catalog # 2498-SE)
Activator: Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
Deactivator: EDTA (Sigma, Catalog # E-4884)
Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH₂ (Catalog # ES002)
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Dilute rmKLKB1 to 200 µg/mL in Activation Buffer.
Dilute Thermolysin to 20 µg/mL in Activation Buffer.
Activate rmKLKB1 by mixing equal volumes of diluted rmKLKB1 and Thermolysin together and incubate at 37 °C for 1 hour.
After incubation, stop reaction with 50 mM EDTA (final concentration).
Dilute incubated rmKLKB1 to 1 ng/µL in Assay Buffer.
Dilute Substrate in Assay Buffer to 20 µM.
In a plate load 50 µL of the 1 ng/µL rmKLKB1, and start the reaction by adding 50 µL 20 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL 20 uM Substrate without any rmKLKB1.
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 V_max* (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).

Per Well:

rmKLKB1: 0.05 µg
Substrate: 10 µM

Reconstitution Calculator

Background: Plasma Kallikrein/KLKB1

Plasma kallikrein, a serine protease, is synthesized in the liver and circulates in the plasma by binding to high molecular weight (HMW) kininogen or as a free zymogen. Once activated by its physiological activator, factor XII, it displays endopeptidase activity towards peptide bonds after arginine (preferred) and lysine. It cleaves HMW kininogen, its major physiological substrate, to release the potent vasodilator peptide bradykinin. It is also able to cleave a number of inactive precursor proteins to generate active products, such as plasminogen and prourokinase. Thus, it plays an important role in blood pressure regulation, fibrinolysis, and neutrophil activation (1). Mouse plasma kallikrein precursor contains a signal peptide (residues 1 to 19) and a pro form sequence (residues 20 to 638). Upon activation, the pro form is converted to a heavy chain and a light chain, which is linked by disulfide bonds and the latter contains the catalytic domain (2). The mouse plasma kallikrein pro form was expressed in the NS0 cells with a foreign signal peptide. After being treated by thermolysin, the purified enzyme is active against a fluorogenic peptide substrate described in the Activity Assay Protocol.

References

Colman, R. (2004) in Handbook of Proteolytic Enzymes, Barrett, A.J. et al. eds. p. 1644.
Seidah, N. et al. (1990) DNA and Cell Biology 9:737.

Entrez Gene IDs

3818 (Human); 16621 (Mouse)

Alternate Names

EC 3.4.21; EC 3.4.21.34; Fletcher factor; kallikrein B, plasma (Fletcher factor) 1; kininogenin; KLK3plasma kallikrein; KLKB1; plasma kallikrein heavy chain; plasma kallikrein light chain; Plasma Kallikrein; Plasma Prekallikrein; PPK

Related Research Areas

Citation for Recombinant Mouse Plasma Kallikrein/KLKB1 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