Recombinant Human Trypsin 2/PRSS2 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.
3586-SE
| 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.
|
Assay Procedure
- Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Assay Buffer: 0.1 M Tris, 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij-35 (w/v), pH 8.0
- Recombinant Human Enteropeptidase/Enterokinase (rhEnterokinase) (Catalog # 1585-SE)
- Recombinant Human Trypsin 2/PRSS2 (rhTrypsin 2) (Catalog # 3586-SE)
- Bacterial Thermolysin (Thermolysin) (Catalog # 3097-ZN)
- 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
- Substrate II: MCA-Arg-Pro-Lys-Pro-Val-Glu-Nva-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 rhEnterokinase to 100 µg/mL in Activation Buffer.
- Dilute Thermolysin to 3.16 µg/mL in Activation Buffer.
- Mix 50 µL of 100 µg/mL rhEnterokinase with 50 µL of 3.16 µg/mL Thermolysin.
- Incubate reaction at 37 °C for 30 minutes.
- Stop reaction by adding 100 µL of 20 mM 1, 10 Phenanthroline to the reaction.
- Dilute activated rhEnterokinase to 0.4 µg/mL in Assay Buffer.
- Dilute rhTrypsin 2 to 200 µg/mL in Assay Buffer.
- Mix 25 µL of 0.4 µg/mL rhEnterokinase with 25 µL of 200 µg/mL rhTrypsin 2.
- Incubate reaction at room temperature for 30 minutes.
- Dilute activated rhTrypsin 2 to 0.05 µg/mL in Assay Buffer.
- Dilute substrate to 20 µM in Assay Buffer.
- In a plate load 50 µL of 0.05 μg/mL of rhTrypsin 2 and include a Substrate Blank of 50 µL Assay Buffer.
- 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.
- 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).
Per Well:- rhTrypsin 2: 0.0025
- Substrate: 10 μM
Reconstitution Calculator
Background: Trypsin 2/PRSS2
Human Trypsin 2, encoded by the PRSS2 gene, is also known as anionic Trypsin (1). Accounting for approximately 1/3 of the total trypsin content in the pancreatic juice, it is one of the trypsins produced in the pancreas. It consists of a signal peptide (residues 1‑15), a pro region (residues 16‑23), and a mature chain (residues 24‑247). Trypsin 2 is synthesized as a zymogen and secreted into the duodenal lumen, where it is activated by enteropeptidase (2). It plays a central role in digestion and activating other pro-enzymes. In human pancreatic diseases and chronic alcoholism, it is up-regulated (3).
- Emi, M. et al. (1986) Gene. 41:305.
- Halfon, S. et al. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, et al.) pp. 1483-1488, Academic Press, San Diego.
- Rinderknecht, H. et al. (1979) Gut. 20:886.
FAQs
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Average Rating: 5 (Based on 1 Review)
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Reason for Rating: We have used this IRT-2 to prepare dried blood spot Standards and Controls. These DBS material is used in our multiplex assay (Luminex xMAP Platform) to screen newborn babies for Cystic Fibrosis.
We started with only IRT-2 and then added a combination of IRT-1 + IRT-2. Both with excellent results.
