Recombinant Human Serpin B2/PAI-2 Protein, CF

R&D Systems | Catalog # 9206-PI

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Human Serpin B2/PAI-2 Protein (9206-PI)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

P05120

Applications

Enzyme Activity
View all Serpin B2 Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human Serpin B2 protein
Glu2-Ala65/Ala99-Pro415, with an N-terminal Met and 6-His tag

Purity

>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Met

Predicted Molecular Mass

44 kDa

SDS-PAGE

41 kDa, reducing conditions

Activity

Measured by its ability to inhibit uPA cleavage of a peptide substrate, N-carbobenzyloxy-Gly-Gly-Arg-7-amido-4-methylcoumarin (Z-GGR-AMC).

The inhibition IC50 is <15 nM, as measured under the described conditions.

Formulation, Preparation, and Storage

9206-PI
Formulation Supplied as a 0.2 μm filtered solution in Sodium Acetate, NaCl and Chaps.
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.

Background: Serpin B2

Serpin B2, also known as PAI-2, is an approximately 60 kDa serine protease inhibitor (1). It is primarily secreted by macrophages and monocytes (2, 3) and can form disulfide-linked multimers (4, 5). Serpin B2 inhibits both the urokinase-type and tissue-type plasminogen activators (uPA and tPA) (3, 4, 6). Serpin B2 also promotes the clearance of uPA by enhancing its binding and uptake by LRP (7). It limits fibril formation by Huntington protein (HTT) and beta-Amyloid peptides (8). It promotes Th2 biased immune responses and is important for intestinal CCL2 production, monocyte recruitment, and nematode clearance (2, 9). A non-glycosylated form of Serpin B2 is retained intracellularly where it interferes with TNF-a induced apoptosis by protecting the Retinoblastoma protein (RB1) from calpain digestion (10). It also inhibits proteasome activity in activated endothelial cells (11). Serpin B2 is upregulated in HIV-1 infected PBMC, induces RB1 upregulation, and promotes HIV-1 replication (12). Human Serpin B2 shares 76% and 73% amino acid sequence identity with mouse and rat Serpin B2, respectively.

References

  1. Shea-Donohue, T. et al. (2014) Gut Microbes 5:254.
  2. Schroder, W.A. et al. (2010) J. Immunol. 184:2663.
  3. Ritchie, H. et al. (1999) Thomb. Haemost. 81:96.
  4. Mikus, P. et al. (1993) Eur. J. Biochem. 218:1071.
  5. Wilczynska, M. et al. (2003) EMBO J. 22:1753.
  6. Baker, M.S. et al. (1990) Cancer Res. 50:4676.
  7. Croucher, D. et al. (2006) J. Biol. Chem. 281:10206.
  8. Lee, J.A. et al. (2015) PLoS One 10:e0130136.
  9. Zhao, A. et al. (2013) J. Immunol. 190:5779.
  10. Tonnetti, L. et al. (2008) Cancer Res. 68:5648.
  11. Boncela, J. et al. (2011) J. Biol. Chem. 286:43164.
  12. Darnell, G.A. et al. (2006) J. Biol. Chem. 281:31348.

Alternate Names

PAI2, PLANH2, Urokinase Inhibitor

Entrez Gene IDs

5055 (Human); 18788 (Mouse); 60325 (Rat)

Gene Symbol

SERPINB2

UniProt

P05120

Additional Serpin B2 Products

Product Documents for Recombinant Human Serpin B2/PAI-2 Protein, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human Serpin B2/PAI-2 Protein, CF

For research use only

Citations for Recombinant Human Serpin B2/PAI-2 Protein, CF

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Protocols

View specific protocols for Recombinant Human Serpin B2/PAI-2 Protein, CF (9206-PI):

Materials
  • Assay Buffer: 50 mM Tris, 0.01% Tween® 20, pH 8.5
  • Recombinant Human Serpin B2 (rhSerpin B2) (Catalog # 9206-PI)
  • Recombinant Human u-Plasminogen Activator (uPA)/Urokinase (rhuPA) (Catalog # 1310-SE)
  • Substrate: Z-Gly-Gly-Arg-AMC (Bachem, Catalog # I-1140), 10 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent
  1. Prepare a curve of rhSerpin B2 (MW: 43.8 kDa) in Assay Buffer. Make the following serial dilutions: 400, 200, 100, 50, 25, 12.5, 6.25, 3.125, and 0.3125 nM. 
  2. Dilute rhuPA to 2 μg/mL in Assay Buffer.
  3. Combine equal volumes of each point of the rhSerpin B2 curve with 2 µg/mL rhuPA. Include an enzyme control containing equal volumes of Assay Buffer and rhuPA.
  4. Incubate reaction mixtures at room temperature for 15 minutes.
  5. Dilute Substrate to 200 µM in Assay Buffer.
  6. Load 50 µL of the diluted 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.
  7. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  8. Derive the 50% inhibition concentration (IC50) value for rhSerpin-B2 by plotting RFU/min (or specific activity) versus concentration with 4-PL fitting.
  9. Calculate specific activity for 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-B2: 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, and 0.078 nM
  • rhuPA: 0.050 µg
  • Substrate: 100 µM

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