Recombinant Mouse Serpin A12 Protein, CF Summary
Leu20-Gly413, with substitutions at Q183R and V403I and a C-terminal 10-His tag
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.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris and NaCl.|
|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.
- Activation Buffer: 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% (w/v) Brij-35, pH 7.5 (TCNB)
- Inhibition Buffer: 25 mM Tris, 150 mM NaCl, pH 7.5
- Assay Buffer: 50 mM Tris, 150 mM NaCl, pH 8.5
- Recombinant Mouse Serpin A12 (rmSerpin A12) (Catalog # 8338-PI)
- Recombinant Human Kallikrein 7 (rhKLK7) (Catalog # 2624-SE)
- Bacterial Thermolysin (Catalog # 3097-ZN)
- 1,10 Phenanthroline (Sigma, Catalog # 320056), 0.6 M stock in DMSO
- Substrate: Mca-RPKPVE-Nval-WRK(Dnp)-NH2 (Catalog # ES002)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent
- Combine rhKLK7 with Thermolysin in Activation Buffer for final concentrations of 100 µg/mL and 10 µg/mL, respectively.
- Incubate rhKLK7 mixture at 37 °C for 2 hours.
- Add 1,10 Phenanthroline at a final concentration of 10 mM to stop activation reaction.
- Prepare a curve of rmSerpin A12 (MW = 47016 Da) in Inhibition Buffer. Make the following serial dilutions: neat, 8000, 4000, 2000, 1000, 500, 250, 100, and 25 nM. (Note: High points may not be achievable due to the stock concentration of some lots).
- Dilute activated/stopped rhKLK7 to 50 μg/mL in Inhibition Buffer.
- Combine equal volumes of each point of the rmSerpin A12 curve with 50 µg/mL rhKLK7. Include an enzyme control containing equal volumes of Inhibition Buffer and 50 µg/mL rhKLK7.
- Incubate curve reaction mixtures at 37°C for 30 minutes.
- Dilute each point of the curve 12.5 fold using Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL each of the diluted curve points to a plate, and start the reactions by adding 50 µL of 20 µM Substrate. Include a Substrate Blank containing 50 µL of Assay Buffer and 50 µL of 20 µM Substrate.
- Read at excitation and emission wavelengths of 320 nm and 405 nm (top read), respectively, in kinetic mode for 5 minutes.
- Derive the 50% inhibition concentration (IC50) value for rmSerpin A12 by plotting RFU/min (or specific activity) versus concentration with 4-PL fitting.
- The specific activity for rhKLK7 at each point may be determined using the following formula:
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-Leu-OH (Bachem, Catalog # M-1975).
- rmSerpin A12: (neat/50), 160, 80, 40, 20, 10, 5, 2, and 0.5 nM
- rhKLK7: 0.1 µg
- Substrate: 10 µM
Background: Serpin A12
Serpin A12, also known as Vaspin, is a 45-50 kDa secreted adipokine that contributes to the maintenance of insulin sensitivity (1, 2). It is structurally related to the Serpin family of serine protease inhibitors (3). Mature mouse Serpin A12 shares 61% and 88% amino acid sequence identity with human and rat Serpin A12, respectively (3). It is expressed by adipocytes in visceral and subcutaneous fat, in the gastric glands and epithelium, and in the placenta (3-5). Serpin A12 circulates in a complex with Kallikrein 7, and it prevents the Kallikrein 7 mediated cleavage of Insulin (6). It promotes the elevation of circulating insulin and improves glucose tolerance but can also inhibit the high glucose induced activation of the Insulin Receptor (3, 6, 7). Serpin A12 inhibits TRANCE/RANK L induced osteoclast development and the inflammatory activation of vascular smooth muscle and endothelial cells (7-9). It additionally functions as an anti-apoptotic protein in vascular endothelial cells and osteoblasts (10, 11).
- Choi, S.H. et al. (2013) Front. Endocrinol. (Lausanne) 4:97.
- Goktas, Z. et al. (2013) Front. Endocrinol. (Lausanne) 4:69.
- Hida, K. et al. (2005) Proc. Natl. Acad. Sci. USA 102:10610.
- Lee, J.A. et al. (2011) Endocr. J. 58:639.
- Caminos, J.E. et al. (2009) Histol. Histopathol. 24:979.
- Heiker, J.T. et al. (2013) Cell Mol. Life Sci. 70:2569.
- Li, H. et al. (2013} Atherosclerosis 228:61.
- Kamino, N. et al. (2013) Connect. Tissue Res. 54:147.
- Jung, C.H. et al. (2014) Cardiovasc. Diabetol. 13:41.
- Jung, C.H. et al. (2011) Biochem. Biophys. Res. Commun. 413:264.
- Zhu, X. et al. (2013) Amino Acids 44:961.
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Fluorogenic Peptide Substrates
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