Recombinant Human Marapsin/Pancreasin Protein, CF
Recombinant Human Marapsin/Pancreasin Protein, CF Summary
Product Specifications
Ala23-Arg34, and Met35-Lys290 with a C-terminal 10-His tag
Analysis
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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.
1988-SE
| Formulation | Lyophilized from a 0.2 μm filtered solution in MES and NaCl. |
| Reconstitution | Reconstitute at 100 μg/mL in sterile 25 mM MES and 100 mM NaCl, pH 6.5. |
| Shipping | The product is shipped at ambient temperature. 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
- Assay Buffer: 50 mM Tris, 250 mM NaCl, 0.05% (w/v) Brij-35, pH 8.0
- Recombinant Human Marapsin/Pancreasin (rhMarapsin) (Catalog # 1988-SE)
- Substrate: Z-Gly-Arg-SBzl (MP Biomedicals, Catalog # SB007)
- 5,5’-Dithio-bis(2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130)
- 96 well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhMarapsin to 0.4 ng/µL in Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer with 200 µM of DTNB.
- Load 50 µL of the diluted rhMarapsin into the plate, and start the reaction by adding 50 µL of the Substrate/DTNB mixture. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL Substrate mixture.
- Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/M |
| ext. coeff.** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Using the extinction coefficient 13260 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD.
Per Well:- rhMarapsin: 0.020 µg
- DTNB: 100 µM
- Substrate: 100 µM
Reconstitution Calculator
Background: Marapsin/Pancreasin
Marapsin, pancreasin, and channel-activating protease 2 (CAP-2) are different names for the same serine protease that is expressed strongly in the pancreas (1, 2). The protein is synthesized with a signal peptide (amino acid residues 1-22), a pro peptide (residues 23-34) and a mature chain (residues 35-290) corresponding to the serine protease domain. The full-length protein was expressed and the secreted protein purified. The N-terminal sequencing results indicate that the purified protein is a disulfide bond-linked dimer formed between the pro peptide and the mature chain. The active enzyme has low activity against peptide substrates tested, but high activity against thioester substrates. The peptidase activity is inhibited by 20 mM benzamidine.
- Bhagwandin, V.J. et al. (2003) J. Biol. Chem. 278:3363.
- Edwards, K.M. et al. (1999) J. Biol. Chem. 274:30468.
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