Recombinant Human Serpin B2/PAI-2 Protein, CF Summary
Product Specifications
The inhibition IC50 is <15 nM, as measured under the described conditions.
Glu2-Ala65/Ala99-Pro415, with an N-terminal Met and 6-His tag
Analysis
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.
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.
|
Assay Procedure
- 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
- 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.
- Dilute rhuPA to 2 μg/mL in Assay Buffer.
- 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.
- Incubate reaction mixtures at room temperature for 15 minutes.
- Dilute Substrate to 200 µM in Assay Buffer.
- 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.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
- Derive the 50% inhibition concentration (IC50) value for rhSerpin-B2 by plotting RFU/min (or specific activity) versus concentration with 4-PL fitting.
- 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)
- 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
Reconstitution Calculator
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.
- Shea-Donohue, T. et al. (2014) Gut Microbes 5:254.
- Schroder, W.A. et al. (2010) J. Immunol. 184:2663.
- Ritchie, H. et al. (1999) Thomb. Haemost. 81:96.
- Mikus, P. et al. (1993) Eur. J. Biochem. 218:1071.
- Wilczynska, M. et al. (2003) EMBO J. 22:1753.
- Baker, M.S. et al. (1990) Cancer Res. 50:4676.
- Croucher, D. et al. (2006) J. Biol. Chem. 281:10206.
- Lee, J.A. et al. (2015) PLoS One 10:e0130136.
- Zhao, A. et al. (2013) J. Immunol. 190:5779.
- Tonnetti, L. et al. (2008) Cancer Res. 68:5648.
- Boncela, J. et al. (2011) J. Biol. Chem. 286:43164.
- Darnell, G.A. et al. (2006) J. Biol. Chem. 281:31348.
Citation for Recombinant Human Serpin B2/PAI-2 Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
1 Citation: Showing 1 - 1
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Cell-Type-Specific Gene Regulatory Networks Underlying Murine Neonatal Heart Regeneration at Single-Cell Resolution
Authors: Z Wang, M Cui, AM Shah, W Tan, N Liu, R Bassel-Dub, EN Olson
Cell Reports, 2020-12-08;33(10):108472.
Species: Human
Sample Types: Whole Cells
Applications: Bioassay
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