Recombinant Mouse Trypsin 3/PRSS3 Protein, CF Summary
Phe16-Asn246, with a C-terminal 10-His tag
Accession # NP_035775
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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||Supplied as a 0.2 μm filtered solution in HCl and NaCl.|
|Shipping||The product is shipped with dry ice or equivalent. 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, 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij-35 (w/v), pH 7.5 (TCNB)
- Assay Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij-35 (w/v), pH 8.0
- Recombinant Mouse Trypsin 3/PRSS3 (rmTrypsin 3) (Catalog # 3565-SE)
- Recombinant Human Enteropeptidase/Enterokinase (rhEnterokinase) (Catalog # 1585-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- 1,10 Phenanthroline (Sigma, Catalog # 320056) 0.6 M in DMSO
- 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
- Activate rhEnterokinase with Thermolysin.
- Dilute rhEnterokinase to 100 µg/mL in Activation Buffer.
- Dilute Thermolysin to 3.16 µg/mL in Activation Buffer.
- Mix equal volumes of diluted rhEnterokinase and Thermolysin.
- Incubate at 37 °C for 30 minutes.
- Stop the reaction by adding an equal volume of 20 mM 1,10 Phenanthroline to the reaction tube.
- Activate rmTrypsin 3 with activated rhEnterokinase.
- Dilute activated rhEnterokinase to 2 µg/mL in Assay Buffer.
- Dilute rmTrypsin 3 to 200 µg/mL in Assay Buffer.
- Mix equal volumes of activated rhEnterokinase and rmTrypsin 3.
- Incubate at room temperature for 1 hour.
- Dilute activated rmTrypsin3 to 0.04 µg/mL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of 0.04 µg/mL of rhTrypsin 3 into a plate, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 20 µM Substrate.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively in kinetic mode for 5 minutes.
- Calculate specific activity:
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).
- rmTrypsin 3: 0.002 µg
- Substrate: 10 µM
Background: Trypsin 3/PRSS3
Mouse Trypsin-3, encoded by the PRSS3 gene, is also known as mesotrypsin. It consists of a signal peptide (residues 1 to 15), a pro region (residues 16 to 23), and a mature chain (residues 24 to 246) (1). The purified recombinant mouse Trypsin 3 corresponds to the pro form, which can be activated by enterokinase.
- Halfon, S. et al. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, A.J. et al.) pp. 1483, Academic Press, San Diego.
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Fluorogenic Peptide Substrates
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