Recombinant Human Serpin A4/Kallistatin Protein, CF
Recombinant Human Serpin A4/Kallistatin Protein, CF Summary
Gln21-Pro427, 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 MES and NaCl.|
|Reconstitution||Reconstitute at 100 μg/mL in sterile 50 mM Tris, 10 mM CaCl2 and 150 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.
- Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, pH 7.5 (TCN)
- Inhibition Buffer: 25 mM Tris, 150 mM NaCl, pH 7.5
- Assay Buffer: 50 mM CHES, 250 mM NaCl, pH 10.0
- Recombinant Human Serpin A4/Kallistatin (rhSerpin A4) (Catalog # 1669-PI)
- Recombinant Human Kallikrein 1 (rhKLK1) (Catalog # 2337-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- 1,10-Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
- Substrate: Pro-Phe-Arg-7-amido-4-methylcoumarin (Bachem, Catalog # I-1295), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Activate rhKLK1 at 100 µg/mL with 1 µg/mL Thermolysin in Activation Buffer.
- Incubate at 37 °C for 1 hour.
- Add 1,10-Phenathroline at a final concentration of 10 mM to stop activation reaction.
- Prepare a curve of rhSerpin A4 (47710 Da) in Inhibition Buffer. Make the following serial dilutions: 8000, 4000, 2000, 1000, 625, 415, 200, and 75 nM.
- Dilute activated rhKLK1 to 5 µg/mL in Inhibition Buffer.
- Combine 20 µL of 5 µg/mL rhKLK1 with 20 µL of rhSerpin A4 curve dilutions. Include two controls of 20 µL Inhibition Buffer with 20 µL of 5 µg/mL rhKLK1.
- Incubate curve at 37 °C for 1 hour.
- Dilute curve 12.5 fold by adding 460 µL of Assay Buffer to each dilution.
- Dilute Substrate to 200 µM in Assay Buffer.
- In a plate load 50 µL of the rhSerpin A4 curve, and start the reaction by adding 50 µL of 200 µM Substrate to wells.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
- Derive the 50% inhibiting concentration (IC50) of rhSerpin A4 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
- The specific activity for rhKLK1 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 (Sigma, Catalog # A-9891).
- rhKLK1: 0.010 µg
- rhSerpin A4 curve: 160, 80, 40, 20, 12.5, 8.3, 4 and 1.5 nM
- Substrate: 100 µM
Background: Serpin A4/Kallistatin
Serpin A4 is a member of the Serpin superfamily of the serine protease inhibitors (1). Known also as kallistatin or proteinase inhibitor 4, Serpin A4 has been shown to function as a specific inhibitor of tissue kallikrein and as an inhibitor of angiogenesis and tumor growth (2, 3).
- Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
- Chen, V.C. et al. (2000) J. Biol. Chem. 275:40371.
- Miao, R.Q. et al. (2002) Blood 100:3245.
Citation for Recombinant Human Serpin A4/Kallistatin 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
Enzymatic properties of human kallikrein-related peptidase 12 (KLK12).
Authors: Memari</LastName><ForeNam N</Initial, Memari N, Jiang W, Diamandis EP, Luo LY
Biol. Chem., 2007;388(4):427-35.
Sample Types: Recombinant Protein
Applications: Enzyme Assay
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