Recombinant Mouse Kininogen HMW (HKa) Protein, CF Summary
Glu21-Ser661, with a C-terminal 10-His tag
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.
|Formulation||Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.|
|Reconstitution||Reconstitute at 100 μg/mL in sterile 25 mM Tris and 150 mM NaCl, pH 7.5.|
|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.
- Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
- Assay Buffer: 50 mM Tris, pH 7.0
- Recombinant Mouse Kininogen High Molecular Weight (HKa) (rmKininogen) (Catalog # 2206-PI)
- Papain (Sigma, Catalog # P4762)
- Substrate: Z-Phe-Arg-AMC (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
- Dilute Papain to 100 µg/mL in Activation Buffer.
- Incubate at room temperature for 10 minutes.
- Prepare a dilution curve of rmKininogen (MW: 72,313 Da) in Assay Buffer. Make the following serial dilutions: 1000, 500, 125, 62.5, 41.7, 27.8, 18.5, 9.26, 3.09, and 1.03 nM.
- Dilute activated Papain to 2.4 µg/mL in Assay Buffer.
- Mix equal volumes of the rmKininogen 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.
- Perform a 1/5 dilution with Assay Buffer to the incubated mixtures.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load 50 µL of diluted incubated mixture into a 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% inhibiting concentration (IC50) value for rmKininogen 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 Vmax* (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).
- Papain: 0.012 µg
- Substrate: 100 µM
- rmKininogen curve: 50, 25, 6.25, 3.125, 2.09, 1.39, 0.925, 0.463, 0.155, and 0.052 nM
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 mouse HMW form is synthesized as a 661 amino acid residue precursor with a signal peptide (residues 1-20). The mature chain (residues 21-661) is further processed into the heavy (residues 21‑379) and the light (residues 389-661) chains. The active peptide bradykinin (residues 380-388) 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 (432 residues) has the same sequence in its heavy chain and bradykinin, but a different sequence in its light chain (residues 401‑432). The LMW form is not involved in blood clotting.
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Fluorogenic Peptide Substrates
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