Recombinant Human Serpin B3/SSCA1 Protein, CF

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

Recombinant Human Serpin B3/SSCA1 Protein, CF Summary

• Details of Functionality: Measured by its ability to inhibit active Cathepsin L cleavage of a fluorogenic peptide substrate Z-LR-AMC (Catalog # ES008). The IC₅₀ is <5 nM, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Serpin B3/SCCA1 protein
  Asn2-Pro390, with an N-terminal Met and 6-His tag
• Accession #: P29508
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: SERPINB3
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 45 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: 40-43 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Assay Procedure:
  • Assay Buffer: 50 mM MES, 5 mM DTT, pH 6.0
  • Recombinant Human Serpin B3/SCCA1 (rhSerpin B3) (Catalog # 6528-PI)
  • Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
  • Substrate: Z-Leu-Arg-AMC (Catalog # ES008)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhCathepsin L to 40 µg/mL in Assay Buffer.
  2. Incubate 40 µg/mL rhCathepsin L for 15 minutes on ice to activate.
  3. Dilute activated rhCathepsin L to 0.2 µg/mL in Assay Buffer.
  4. Prepare a curve of rhSerpin B3 (MW = 45386 Da) in Assay Buffer. Make the following serial dilutions: 1000, 200, 50, 35, 25, 15, 5, 2 and 1 nM.
  5. Combine 25 µL of each dilution with 25 µL of the 0.2 µg/mL rhCathepsin L. Include an enzyme control containing Assay Buffer in place of rhSerpin B3.
  6. Incubate reaction mixtures at 37 °C for 15 minutes.
  7. Dilute reactions by adding 200 µL Assay Buffer to each.
  8. Dilute Substrate to 20 µM in Assay Buffer.
  9. Load into a black well plate 50 µL of the diluted incubated mixtures and start the reaction by adding 50 µL substrate.
  10. Read at excitation and emission wavelengths of 380 nm and 460 nM (top read), respectively, in kinetic mode for 5 minutes.
  11. Derive the 50% inhibition concentration (IC₅₀) value for rhSerpin B3 by plotting RFU/min vs. concentration with 4-PL fitting.
  12. Calculate the specific activity for rhCathepsin L at each point using the following formula (if needed):

  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 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).

  Per Well:
  • rhCathepsin L: 0.001 µg
  • rhSerpin B3: 50, 10, 2.5, 1.75, 1.25, 0.75, 0.25, 0.1, and 0.05 nM
  • Substrate: 10 μM

Notes

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

Alternate Names for Recombinant Human Serpin B3/SSCA1 Protein, CF

• HsT1196
• Protein T4-A
• SCC
• SCCA
• SCCA1
• SCCA1SCCA-1
• SCCA-PD
• serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 3
• Serpin B3
• serpin peptidase inhibitor, clade B (ovalbumin), member 3
• Squamous cell carcinoma antigen 1
• T4-A

Background

Serpin B3, also known as squamous cell carcinoma antigen-1 (SCCA-1), is a member of the serpin superfamily of serine protease inhibitors (1). Serpins primarily inhibit serine proteases and some are known to inhibit caspases and papain-like cysteine proteases (1). Serpin B3 belongs to the subgroup ovalbumin-related serpins which are involved in the regulation of apoptosis, inflammation, angiogenesis and embryogenesis (2). Serpin B3 was initially isolated from human cervical squamous cell tissue. It is a tumor marker for squamous cell carcinomas in various areas including the cervix, head, neck and lung (3). Unlike typical serpins, Serpin B3 shows inhibitory activity against papain-like lysosomal cysteine proteases Cathepsin K, L, and S, while lacking inhibitory activity against serine proteases (4).

1. Law, R. et al. (2006) Genome Biol. 7:216.
2. Benarafa, C. and Remold-O’Donnell, E. (2005) Proc. Natl. Acad. Sci. 102:11367.
3. Higgens, W.J. et al. (2010) J. Biol. Chem. 285:3722.
4. Schick, C. et al. (1998) Biochemistry. 37:5258.

Bioinformatics

• Gene Symbol: SERPINB3
• Uniprot:
  • P29508()