Recombinant Human Kininogen HMW (HKa) Protein, CF

Catalog #: 1569-PI
1 Citation Datasheet / COA / SDS

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1569-PI-010

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

Recombinant Human Kininogen HMW (HKa) 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 inhibit papain cleavage of a fluorogenic peptide substrate Z-FR-AMC (Catalog # ES009). The IC₅₀ value is <3 nM, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived human Kininogen protein
Gln19-Ser644, with an N-terminal signal sequence and a C-terminal 10-His tag

Accession #

P01042

N-terminal Sequence
Analysis

Ser390 (light chain) & Lys438 (minor species) with Gln19 predicted to be present and blocked

Structure / Form

High molecular weight form consisting of the mature chain, the heavy and light chains, and a minor species

Predicted Molecular Mass

71 kDa (mature), 41 kDa (heavy) & 30 kDa (light)

SDS-PAGE

120 kDa, 60 kDa and 48 kDa, reducing conditions

Product Datasheets

1569-PI

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.

1569-PI

Formulation	Lyophilized from a 0.2 μm filtered solution in Sodium Acetate and NaCl.
Reconstitution	Reconstitute at 100 μg/mL in sterile 25 mM Tris and 100 mM NaCl, pH 7.5.
Shipping	The product is shipped at ambient temperature. 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 reconstitution.

Assay Procedure

Materials

Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
Assay Buffer: 50 mM Tris, pH 7.0
Recombinant Human Kininogen High Molecular Weight (HKa) (rhKininogen) (Catalog # 1569-PI)
Papain (Sigma, Catalog # P4762)
Substrate: Z-Phe-Arg-AMC (R&D Systems, Catalog # ES009), 10 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

Chill Activation Buffer on ice.
Dilute Papain to 100 µg/mL in Activation Buffer.
Incubate at room temperature for 10 minutes.
Prepare a dilution curve of rhKininogen (MW: 71,211Da) in Assay Buffer. Make the following serial dilutions: 400, 200, 100, 50, 25, 12.5, 6.25, and 3.125 nM.
Dilute activated Papain to 2 µg/mL in Assay Buffer.
Mix equal volumes of the rhKininogen curve dilutions and the diluted active Papain. Include a control (in duplicate) containing Assay Buffer and the diluted active Papain.
Incubate mixtures at room temperature for 10 minutes.
Dilute Substrate to 200 µM in Assay Buffer.
Perform a 1/5 dilution with Assay Buffer to the incubated mixture of rhKininogen and Papain.
Load 50 µL of diluted incubated mixture into the plate, and start the reaction by adding 50 µL of 200 µM Substrate.
Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes in kinetic mode.
Derive the 50% inhibition concentration (IC₅₀) for rhKininogen by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
The specific activity for Papain at each point may be determined using the following formula (if needed):

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 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891).

Per Well:

Papain: 0.010 µg
Substrate: 100 µM
rhKininogen curve: 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 nM

Reconstitution Calculator

Background: Kininogen

Kininogen, also known as alpha 2-thiol proteinase inhibitor, is a multi-function protein. There are two alternatively spliced forms, designated as the high molecular weight (HMW) and low MW (LMW) forms (1). The HMW form is synthesized as a 644 amino acid residue precursor with a signal peptide (residues 1‑18). The mature chain (residues 19‑644) is further processed into the heavy (residues 19‑380) and the light (residues 390‑644) chains. The active peptide bradykinin (residues 381‑389) is released, which has a variety of functions including muscle contraction, hypotension and inflammation. The heavy chain consists of three cystatin‑like domains, which are responsible for inhibiting cysteine proteases. The light chain consists of a His-rich domain, which is associated with the clotting activity. In comparison to the HMW form, the LMW Kininogen (427 residues) has the same sequence in its heavy chain and bradykinin, but a different sequence in its light chain (residues 402‑427). The LMW form is not involved in blood clotting.

References

Takagaki, Y. et al. (1985) J. Biol. Chem. 260:8601.

Entrez Gene IDs

3827 (Human); 16644 (Mouse); 24903 (Rat)

Alternate Names

alpha 2-thiol Proteinase Inhibitor; BDK; BDK;BK;HAE6;HK;HMWK;Kininogen-1;KNG;KNG1; Kininogen; KNG1

Related Research Areas

Citation for Recombinant Human Kininogen HMW (HKa) 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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