Recombinant Human Serpin B4 Protein, CF

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

Recombinant Human Serpin B4 Protein, CF Summary

• Details of Functionality: Measured by its ability to inhibit chymase cleavage of a fluorogenic peptide substrate, Suc-AAPF-AMC. The IC₅₀ is <20 nM, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Serpin B4 protein
  Asn2-Pro390
  with an N-terminal Met and 6-His tag
• Accession #: P48594
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 46 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: 38-44 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 reconstitution.
• Buffer: Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >90%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Reconstitution Instructions: Reconstitute at 0.5 mg/mL in deionized water.
• Assay Procedure:
  • Maturation Buffer: 50 mM MES, pH 5.5
  • Cathepsin Buffer: 50 mM MES, 50 mM NaCl, 5 mM DTT, pH 5.5
  • Assay Buffer: 20 mM Tris, 2 M KCl, 0.02% (v/v) Triton® X-100, pH 9.0
  • Recombinant Human Serpin B4 (rhSerpin B4) (Catalog # 9437-PI)
  • Recombinant Human Chymase/CMA1 (rhChymase) (Catalog # 4099-SE)
  • Recombinant Mouse Active Cathepsin C/DPPI (rmCathepsin C) (Catalog # 2336-CY)
  • Heparin (Tocris, Catalog # 2812), 20 mg/mL stock in deionized water
  • Substrate: Suc-Ala-Ala-Pro-Phe-AMC (Bachem, Catalog # I-1465), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhChymase-1 to 20 µg/mL in Maturation Buffer.
  2. Dilute rmCathepsin C to 20 µg/mL in Cathepsin Buffer.
  3. Dilute Heparin to 2.5 mg/mL in deionized water.
  4. Combine equal volumes of 20 µg/mL rhChymase and 20 µg/mL rmCathepsin.
  5. Add 2.5 mg/mL Heparin to the rhChymase mixture for a final concentration of 49 µg/mL.
  6. Incubate rhChymase mixture at room temperature for 1 hour.
  7. Dilute the rhChymase mixture to 4 µg/mL of rhChymase in Assay Buffer and incubate at room temperature for 5 minutes.
  8. Prepare a curve of rhSerpin B4 (MW: 45,677 Da) in Assay Buffer. Make the following serial dilutions: 2000, 500, 250, 125, 62.5, 31.25, 15.624, 7.812, 3.908 and 0.3908 nM.
  9. Combine equal volumes of each point of the rhSerpin B4 curve with 4 µg/mL rhChymase. Include an enzyme control containing equal volumes of Assay Buffer and 4 µg/mL CMA1.
  10. Incubate reaction mixtures at room temperature for 30 minutes.
  11. Dilute Substrate to 200 µM in Assay Buffer.
  12. Load 50 µL of the incubated mixtures into empty wells of a plate, and start the reaction by adding 50 µL of 200 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 200 µM Substrate.
  13. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  14. Derive the 50% inhibition concentration (IC50) value for rhSerpin B4 by plotting RFU/min (or specific activity) versus concentration with 4-PL fitting.
  15. Calculate specific activity for rhChymase 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 # A9891).

  Per Well:
  rhSerpin B4: 500, 125, 62.5, 31.25, 15.625, 7.812, 3.906, 1.953, 0.977, and 0.0977 nM
  rhChymase-1: 0.1 µg
  Substrate: 100 µM

Notes

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

Alternate Names for Recombinant Human Serpin B4 Protein, CF

• LEUPIN
• Peptidase Inhibitor 11
• PI11
• PI-11
• PI11clade B (ovalbumin), member 4
• SCCA2
• Serpin B4
• serpin peptidase inhibitor, clade B (ovalbumin), member 4
• SerpinB4
• squamous cell carcinoma antigen 1
• Squamous cell carcinoma antigen 2

Background

Serpin B4, also known as squamous cell carcinoma antigen 2 (SCCA-2), is an approximately 45 kDa member of the serpin superfamily of serine protease inhibitors (1). Serpin B4 belongs to the subgroup ovalbumin-related serpins which are involved in the intracellular regulation of apoptosis, inflammation, angiogenesis and embryogenesis (2). Serpin B4 shares 91% sequence identity to Serpin B3, also known as squamous cell carcinoma antigen 1 (SCCA-1), with key differences in the reactive site loops leading to inhibition of distinct classes of proteases (3). Serpin B4 inhibits chymotrypsin-like serine proteases (3). Serpin B4 inhibits apoptosis via STAT activation in an infection (4) and in carcinoma cells (5). It is a mediator in Ras-driven cancer and inflammation (6). Serpin B4 also is a core protein from which Pso p27, an autoantigen present in chronic inflammatory diseases, is derived (7, 8).

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. Schick, C. et al. (1998) Proc. Natl. Acad. Sci. 95:13465.
4. Song, K.J. et al. (2012) Korean J. Parasitol. 50:1.
5. Ahmed, S.T. and Darnell, J.E. (2009) Biochem. Biophys. Res. Commun. 378:821.
6. Catanzaro, J.M. et al. (2014) Cell Cycle 13:3155.
7. Lysvand, H. et al. (2014) Biochim. Biophys. Acta. 1842:734.
8. Iverson, O.J. et al. (2017) Clin. Immunol. 174:10.

Bioinformatics

• Uniprot:
  • P48594()