Recombinant Human Trypsin 1/PRSS1 Protein, CF Summary
Product Specifications
Ala16-Ser247, with a C-terminal 10-His tag
Analysis
Product Datasheets
Carrier Free
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.
3848-SE
| Formulation | Supplied as a 0.2 μm filtered solution in HCl and NaCl. |
| 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.
|
Assay Procedure
- Activation Buffer: 50 mM Tris, 0.15 M NaCl, 10 mM CaCl2, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Assay Buffer: 100 mM Tris, 150 mM NaCl, 10 mM CaCl2, 0.05% (w/v) Brij-35, pH 8.0
- Recombinant Human Trypsin 1/PRSS1 (rhTrypsin 1) (Catalog # 3848-SE)
- Recombinant Human Enteropeptidase/Enterokinase (rhEnterokinase) (Catalog # 1585-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH2 (Catalog # ES002) 2 mM stock in DMSO
- 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Activate rhEnterokinase (see also R&D Systems, Catalog # 1585-SE).
- Activate rhEnterokinase at 50 µg/mL with 1.58 µg/mL Thermolysin in Activation Buffer.
- Incubate at 37 °C for 30 minutes.
- Stop the reaction with an equal volume of 20 mM of 1,10 Phenanthroline for a final concentration of 10 mM.
- Activate rhTrypsin 1 at 100 µg/mL with activated rhEnterokinase at 0.4 µg/mL.
- Dilute the activated rhEnterokinase to 0.8 µg/mL in Assay Buffer.
- Dilute rhTrypsin 1 to 200 µg/mL in Assay Buffer.
- Mix equal volumes of 0.8 µg/mL rhEnterokinase with 200 µg/mL rhTrypsin 1.
- Incubate reaction at room temperature for 15 minutes.
- Dilute activated rhTrypsin 1 to 0.1 µg/mL in Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- In a plate load 50 µL of 0.1 µg/mL rhTrypsin 1 to wells, and start the reaction by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL 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).
- rhTrypsin 1: 0.005 µg
- Substrate: 10 µM
Reconstitution Calculator
Background: Trypsin 1/PRSS1
Human Trypsin 1, encoded by the PRSS1 gene, is also known as cationic trypsinogen (1). Constituting approximately two-thirds of the total trypsin content in normal pancreatic juice, it is the most abundant trypsin isoform produced by the pancreas. It contains a signal peptide (residues 1‑15), a pro region (residues 16‑23), and a mature chain (residues 24‑247). Trypsin 1 is synthesized in the pancreas and secreted into the duodenum lumen, where it is activated by enterokinase. Its major physiologic function is to digest food and to activate other pro-enzymes (2). Mutations in the PRSS1 gene can cause hereditary pancreatitis (3).
- Emi, M. et al. (1986) Gene 41:305.
- Halfon, S. et al. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, et al.) p. 1483, Academic Press, San Diego.
- Teich, N. et al. (2006) Hum. Mutat. 27:721.
FAQs
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