Recombinant Human Serpin F2/alpha 2-Antiplasmin Protein, CF Summary
Details of Functionality
Measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002). The IC50 value is <2.0 nM, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived human Serpin F2/alpha 2-Antiplasmin protein Met28-Lys491, with a C-terminal 10-His tag
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.
Applications/Dilutions
Dilutions
Inhibition Activity
Theoretical MW
53 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
67 kDa, reducing conditions
Publications
Read Publications using 1470-PI in the following applications:
Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH2 (Catalog # ES002), 2 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Dilute Trypsin to 0.25 µg/mL in Assay Buffer and KEEP ON ICE.
Prepare a curve of rhSerpin F2 (MW: 53,056 Da) in Assay Buffer. Make the following serial dilutions: 600, 200, 100, 50, 30, 20, 15, 10, 5 and 1 nM.
Combine equal volumes of 0.25 µg/mL Trypsin and rhSerpin F2 serial dilutions. Include two enzyme controls of equal volumes of Assay Buffer and 0.25 µg/mL Trypsin.
Incubate reaction mixtures at 37 °C for 15 minutes.
Dilute incubated reaction mixtures by 1/5 in Assay Buffer.
Dilute Substrate to 20 µM with Assay Buffer.
In a plate load 50 µL of the diluted reaction mixtures, and start the reaction by adding 50 µL of 20 µM Substrate to wells.
Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
Derive the 50% inhibition concentration (IC50) for rhSerpin F2 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
The specific activity for Trypsin 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 MCA-Pro-Leu-OH (Bachem, Catalog # M-1975).
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Serpin F2/alpha 2-Antiplasmin Protein, CF
AAPclade F (alpha-2 antiplasmin
alpha 2-Antiplasmin
Alpha-2-AP
Alpha-2-PI
Alpha-2-plasmin inhibitor
pigment epithelium derived factor), member 2
Serpin F2
serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epitheliumderived factor), member 2
Background
Serpin F2 is a member of the Serpin superfamily and the primary physiological inhibitor of the serine protease plasmin, which is responsible for the dissolution of fibrin clots (1, 2). In addition to plasmin, Serpin F2 is also an efficient inhibitor of trypsin and chymotrypsin (3). Liver and kidney are major sites of Serpin F2 production and other tissues such as muscle, intestine, central nervous system, and placenta also express its mRNA at a moderate level. The tissue expression pattern of Serpin F2 indicates that it is a key regulator of plasmin-mediated proteolysis in these tissues (4). Human Serpin F2 is synthesized as a 491 amino acid precursor with a 27 amino acid signal peptide. The secreted protein has a short propeptide (residues 28-39) and a mature chain (residues 40-491). The presence of the propeptide did not affect its ability to inhibit plasmin but reduced its cross-linking ability to fibrin (5).
Tone, M. et al. (1987) J. Biochem. 102:1033.
Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
Potempa, J. et al. (1988) Science 241:699.
Menoud, P.-A. et al. (1996) J. Clin. Invest. 97:2478.
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