Recombinant Mouse Serpin E2/PN1 Protein, CF Summary
Product Specifications
Ser20-Pro397, with a C-terminal 10-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.
2175-PI
| Formulation | Supplied as a 0.2 μm filtered solution in Sodium Acetate, NaCl and Glycerol. |
| 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, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Recombinant Mouse Serpin E2/PN1 (rmSerpin E2) (Catalog # 2175-PI)
- Trypsin (Sigma, Catalog # T-1426)
- Substrate: MCA-Arg-Pro-Lys-Pro-Val-Glu-NVAL-Trp-Arg-Lys(DNP)-NH2 (Catalog # ES002)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute Trypsin to 0.25 μg/mL in Assay Buffer and KEEP ON ICE.
- Prepare a curve of rmSerpin E2 (MW: 43,200 Da) in Assay Buffer. Make the following serial dilutions: 500, 100, 50, 30, 20, 10, 5, 2.5, and 0.625 nM.
- Combine equal volumes of diluted Trypsin and rmSerpin E2 at each concentration of the curve. Include two controls containing equal volumes of Assay Buffer and diluted Trypsin.
- Incubate at room temperature for 30 minutes.
- Dilute reaction mixtures five fold in Assay Buffer.
- Dilute Substrate to 20 μM in Assay Buffer.
- Load 50 μL of the diluted mixtures in a plate, and start the reaction by adding 50 μL of 20 μM Substrate to wells.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
- Derive the 50% inhibiting concentration (IC50) of rmSerpin E2 by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
- The specific activity for Trypsin at each point may be determined 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 MCA-Pro-Lys-OH (Bachem, Catalog # M-1975).
Per Well:- Trypsin: 0.00125 μg
- rmSerpin E2 curve: 25, 5, 2.5, 1.5, 1, 0.5, 0.25, 0.125, and 0.03125 nM
- Substrate: 10 μM
Reconstitution Calculator
Background: Serpin E2/PN1
Serpin E2, also known as protease nexin I or glial-derived nexin (GDN), is a member of the Serpin superfamily of the serine protease inhibitors (1). Serpin E2 is a potent inhibitor of thrombin, plasmin and plasminogen activators (2). It is differentially expressed during neuronal differentiation and is able to transform human embryonic kidney cells into neuron-like cells (3). Its over‑expression in mice leads to progressive neuronal and motor dysfunction in these animals (4). It is also over‑expressed in the majority of pancreatic carcinoma as well as gastric and colorectal cancer samples whereas it is weakly expressed in all normal pancreas and chronic pancreatitis tissue samples (5). It plays an important role in controlling male fertility because its knockout male mice show a marked impairment in fertility from the onset of sexual maturity and its abnormal expression is found in the semen of men with seminal dysfunction (6).
- Silverman, G.A. et al. (2001) J. Biol. Chem. 276:33293.
- Rossignol, P. et al. (2004) J. Biol. Chem. 279:10346.
- Lin, H.J. et al. (2005) Int. J. Dev. Neurosci. 23:9.
- Meins, M. et al. (2001) J. Neurosci. 21:8830.
- Buchholz, M. et al. (2003) Cancer Res. 63:4945.
- Murer, V. et al. (2001) Proc. Natl. Acad. Sci. USA 98:3029.
Citation for Recombinant Mouse Serpin E2/PN1 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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Mechanical Loading-Driven Tumor Suppression Is Mediated by Lrp5-Dependent and Independent Mechanisms
Authors: Y Feng, S Liu, R Zha, X Sun, K Li, A Robling, B Li, H Yokota
Cancers, 2021-01-13;13(2):.
Species: Mouse
Sample Types: Whole Cells
Applications: Bioassay
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