Recombinant Mouse Prss28 Protein, CF

• Reactivity: Mu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Mouse Prss28 Protein, CF Summary

• Details of Functionality: Measured by its ability to cleave a peptide substrate, Suc-VPF-SBzl after activation by Thermolysin. The specific activity is >15,000 pmol/min/µg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived mouse PRSS28 protein
  Ala19-Gln274, with a C-terminal 10-His tag
  Accession # Q924N9
• Accession #: Q924N9
• N-terminal Sequence: Ala19
• Structure / Form: Pro form
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Prss28
• Purity: >90%, 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: 30 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: 34 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Activation Buffer: 50 mM Tris, 150 mM NaCl, 10 mM CaCl₂, 0.05% Brij-35, pH 7.5 (TCNB)
  • Assay Buffer: 50 mM Tris, 150 mM NaCl, pH 8.0
  • Recombinant Mouse PRSS28 (rmPRSS28) (Catalog # 4808-SE)
  • Bacterial Thermolysin (Catalog # 3097-ZN)
  • 1,10-Phenanthroline (Sigma, Catalog # 320056), 600 mM stock in DMSO
  • Substrate: SUC-Val-Pro-Phe-SBZL (Bachem, Catalog # M-1885), 10 mM stock in DMSO
  • 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB) (Sigma, Catalog # D-8130), 10 mM stock in DMSO
  • 96 well clear plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rmPRSS28 to 100 µg/mL with 0.1 µg/mL Thermolysin in Activation Buffer.
  2. Incubate at 37 °C for 60 minutes.
  3. Stop Thermolysin activity by adding 1,10-Phenanthroline to 10 mM.
  4. Incubate at room temperature for 15 minutes.
  5. Dilute active rmPRSS8 to 0.05 µg/mL in Assay Buffer.
  6. Dilute Substrate to 200 µM with 200 µM DTNB in Assay Buffer.
  7. Load 50 µL of the 0.05 µg/mL rmPRSS28 into a clear microplate. Include a Substrate Blank containing 50 µL of Assay Buffer.
  8. Start the reaction by adding 50 µL of the Substrate Mixture into wells.
  9. Read in kinetic mode for 5 minutes at an absorbance of 405 nm.
  10. Calculate specific activity using the following formula:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (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 0.320 cm
  Note: the output of many spectrophotometers is in mOD. Per Well:
  • rmPRSS8: 2.5 ng
  • Substrate: 100 µM
  • DTNB: 100 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse Prss28 Protein, CF

• ISP1
• PRSS28

Background

Mouse Prss28, also known as Implantation Serine Protease 1 (ISP1), belongs to the S1 serine proteinase family with a conserved Histidine-Aspartic Acid-Serine catalytic triad (1). It closely resembles the mouse mast cell tryptases, specifically MCP-6 and MCP-7 (1). It is co-expressed with ISP2/Prss29 in pre-implantation embryos and uterus (2). It is involved with embryo hatching and its activity is important for successful implantation (2).

1. Tang, L. and D. E. Rancourt (2005) Gene 364:30.
2. Sharma, N. et al. (2006) BMC Developmental Biology 6:61.
   
   

Bioinformatics

• Gene Symbol: Prss28
• Uniprot:
  • Q924N9()