Measured by its ability to cleave the fluorogenic peptide substrate Boc-QAR-AMC (Catalog # ES014). The specific activity is >15 pmol/min/µg, as measured under the described conditions.
Source
Mouse myeloma cell line, NS0-derived mouse Prostasin/Prss8 protein Ala30-Gln289 with an N-terminal signal sequence and 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
Enzyme Activity
Theoretical MW
29 kDa & 28 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
37-39 kDa, reducing conditions
Publications
Read Publications using 2968-SE in the following applications:
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Dilute rmProstasin to 20 µg/mL in Assay Buffer.
Dilute Substrate to 200 µM in Assay Buffer.
Load into a black well plate 50 µL of 20 µg/mL rmProstasin and start the reaction by adding 50 µL of 200 µM Substrate. As a Substrate Blank combine 50 µL of Assay Buffer and 50 µL of 200 µM Substrate.
Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
Calculate specific activity:
Specific Activity (pmoles/min/µg) =
Adjusted Vmax* (RFU/min) x Conversion Factor** (pmole/RFU)
amount of enzyme (µg)
*Adjusted for Substrate Blank **Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).
Per Well:
rmProstasin: 1 µg
Substrate: 100 µM
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Mouse Prostasin/Prss8 Protein, CF
CAP1
channel-activating protease 1
EC 3.4.21
EC 3.4.21.-
EC 3.4.21.120
Prostasin
protease, serine, 8
Prss8
Serine protease 8
Background
Prostasin encoded by the Prss8 gene, also known as channel activating protease 1, is a serine protease with a trypsin-like substrate specificity (1, 2). The preproenzyme possesses a C-terminal membrane-spanning domain that can be proteolytically processed to generate a secreted form of the enzyme. The secreted form of prostasin has been purified from seminal fluid (2). Prostasin is highly expressed in the prostate gland, and is expressed at lower levels in the lung, kidney, liver, salivary gland, and pancreas (3). The peptidase activity of prostasin is involved in the regulation of epithelial sodium channels (4, 5). Secreted from NS0 cells as the active form, the purified recombinant mouse (rm) Prostasin most likely consists of a disulfide bond-linked two chains with N-terminal sequences of I45TGGGSAKPG and A30DGTE, respectively (3). The enzymatic activity of rmProstasin is effectively inhibited by rmHAI-1 and rmHAI‑2B (Catalog # 1141-PI and 1107-PI).
Vuagniaux, G. et al. (2000) J. Am. Soc. Nephrol. 11:828.
Yu J.X. et al. (1994) J. Biol. Chem. 269:18843.
Yu J.X. et al. (1995) J. Biol. Chem. 270:13483.
Caldwell R.A. et al. (2004) Am. J. Physiol. Cell Physiol. 286:C190.
Tong Z. et al. (2004) Am. J. Physiol. Lung Cell Mol. Physiol. 287:L928.
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