Recombinant Human Serpin A12 Protein, CF

Catalog #: 8337-PI Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
8337-PI-050

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Recombinant Human Serpin A12 Protein, CF Summary

Product Specifications

Purity

>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

Endotoxin Level

<0.10 EU per 1 μg of the protein by the LAL method.

Activity

Measured by its ability to inhibit KLK7 cleavage the fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (Catalog # ES002). The IC₅₀ is <75 nM, as measured under the described conditions.

Source

Mouse myeloma cell line, NS0-derived human Serpin A12 protein
Leu20-Lys414, with C-terminal 10-His tag

Accession #

Q8IW75

N-terminal Sequence
Analysis

Leu20

Predicted Molecular Mass

47 kDa

SDS-PAGE

54-60 kDa, reducing conditions

Product Datasheets

8337-PI

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

8337-PI

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. 6 months from date of receipt, -20 to -70 °C as supplied. 3 months, -20 to -70 °C under sterile conditions after opening.

Assay Procedure

Materials

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 Human Serpin A12 (rhSerpin A12) (Catalog # 8337-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 rhSerpin A12 (MW = 46593 Da) in Inhibition Buffer. Make the following serial dilutions: neat, 8000, 4000, 2000, 1000, 500, 250, and 50 nM. (Note: High points may not be achievable due to the stock concentration of some lots).
Dilute stopped rhKLK7 to 50 μg/mL in Inhibition Buffer.
Combine equal volumes of each point of the rhSerpin 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 room temperature 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 rhSerpin 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 V_max* (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).

Per Well:

rhSerpin A12: (neat/50), 160, 80, 40, 20, 10, 5, and 1 nM
rhKLK7: 0.1 µg
Substrate: 10 µM

Reconstitution Calculator

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 human Serpin A12 shares 61% amino acid sequence identity with mouse and rat Serpin A12 (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).

References

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.

Entrez Gene IDs

145264 (Human); 68054 (Mouse); 191570 (Rat)

Alternate Names

antitrypsin), member 12; member 12; OL-64; OL-64Visceral adipose-specific serpin; serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase; Serpin A12; serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin); Vaspin; Visceral adipose tissue-derived serine protease inhibitor