Recombinant Mouse Serpin A12 (carrier-free)

Product Details

Source

Mouse Serpin A12 (Vaspin), amino acids Gln22-Gly413 (Accession # BC062143) with a C-terminal TG-8H-GGQ tag was expressed in CHO cells.

Molecular Mass

The 406 amino acid recombinant protein has a predicted molecular mass of approximately 47.0 kDa. The non-reduced and DTT-reduced proteins migrate at 50 - 60 kDa by SDS-PAGE.

Purity

>95%, as determined by Coomassie stained SDS-PAGE.

Formulation

0.22 µm filtered protein solution is in pH 7.5 buffer containing 20 mM TRIS and 300 mM NaCl.

Endotoxin Level

Less than 1.0 EU per µg of protein as determined by the LAL method.

Concentration

10 and 25 µg sizes are bottled at 200 µg/mL. 100 µg size and larger sizes are lot-specific and bottled at the concentration indicated on the vial. To obtain lot-specific concentration and expiration, please enter the lot number in our Certificate of Analysis online tool.

Storage & Handling

Unopened vial can be stored between 2°C and 8°C for up to 2 weeks, at -20°C for up to six months, or at -70°C or colder until the expiration date. For maximum results, quick spin vial prior to opening. The protein can be aliquoted and stored at -20°C or colder. Stock solutions can also be prepared at 50 - 100 µg/mL in appropriate sterile buffer, carrier protein such as 0.2 - 1% BSA or HSA can be added when preparing the stock solution. Aliquots can be stored between 2°C and 8°C for up to one week and stored at -20°C or colder for up to 3 months. Avoid repeated freeze/thaw cycles.

Activity

Mouse Serpin A12 (Vaspin) inhibits the peptidase activity of human KLK7 with an IC₅₀ value less than 35 nM (1.65 µg/mL).

Application

Bioassay

Application Notes

Materials and Buffers

1. Assay Buffers: pH 8.5, 50 mM Tris, 0.15 M NaCl, 0.01% Tween-20
2. KLK7 Activation Buffer: pH 7.5, 50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35
3. Activation stop solution: 500 mM EDTA at pH 8.0
4. Human KLK7
5. Mouse Serpin A12 (47.0 kDa)
6. Bacterial Thermolysin (TLN)
7. KLK7 substrate: Mca-RPKPVE-Nval-WRK(Dnp)-NH₂ (2 mM in DMSO)

Assay Procedures

1. Activate at hKLK7 at 100 ug/mL in the presence of thermolysin at 20 ug/mL in KLK7 Activation Buffers. Incubate at 37 °C for 2 hours for activation. Quench the activation by adding equal volume of activation stop solution (500 mM EDTA at pH 8.0). After quenching, hKLK7 concentration is 50 ug/mL.
2. Dilute recombinant mSerpin A12 in Assay Buffer at 200 ug/mL, if it is necessary. Make a serial dilution of following concentration from 200 ug/mL in 20 uL: 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 and 0 ug/mL.
3. Add 5 uL of 50 ug/mL of the activated human KLK7 stock from step 1 to the prepared mSerpin A12 solutions. Briefly centrifuge to mix the solution.
4. Incubate hKLK7 and mSerpin A12 mixture for 1 hour at RT.
5. Prepare 20 uM hKLK7 substrate solution in Assay Buffer.
6. Add 100 uL of Assay Buffer to each hKLK7 and mSerpin A12 mixture.
7. Transfer 50 uL of each mixture from step 6 to a microplate.
8. Start the assay by adding 50 uL of 20 uM hKLK7 substrate solution.
9. Read the reaction progress at 320/405 nm (Excitation/Emmision).

• The Serpin A12 concentration in the final assay is 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0 ug/mL.
• hKLK7 is 1.0 ug/mL (100 ng) in the final assay.
• The substrate concentration is 10 µM in the final assay.

The molar concentration of Serpin A12 was calculated by MW 47.0 kDa.

BioLegend carrier-free recombinant proteins provided in liquid format are shipped on blue-ice. Our comparison testing data indicates that when handled and stored as recommended, the liquid format has equal or better stability and shelf-life compared to commercially available lyophilized proteins after reconstitution. Our liquid proteins are verified in-house to maintain activity after shipping on blue ice and are backed by our 100% satisfaction guarantee. If you have any concerns, contact us at tech@biolegend.com.

Antigen Details

Distribution

Expressed in visceral adipose tissues. Possibly present in adipocytes. Present in the plasma and serum.

Function

Adipokines that modulate insulin action by specifically inhibiting target protease KLK7 in white adipose tissues.

Interaction

KLK7

Ligand/Receptor

KLK7, GRP78/VDAC complex.

Bioactivity

KLK7 inhibition activity

Biology Area

Neuroscience, Stem Cells

Molecular Family

Enzymes and Regulators

Antigen References

1. Hida K, et al. 2005. Proc. Natl. Acad. Sci. USA 30:10610.
2. Li HL, et al. 2011. Clin. Chem. Lab Med. 49:1547.
3. Jung CH, et al. 2011. Biochem. Biophys. Res. Commun. 413:264.
4. Nakatsuka A, et al. 2012. Diabetes 61:2823.
5. Li H, et al. 2013. Atherosclerosis 228:61.
6. Heiker JT, et al. 2013. Cell Mol. Life Sci. 70:2569.
7. Jian W, et al. 2014. PLoS One 9:e94763.

Gene ID

68054 View all products for this Gene ID

UniProt

View information about Serpin A12 on UniProt.org

Documentation

• Technical Data Sheet (pdf)
• Certificate of Analysis
• Safety Data Sheet

Reviews

Related Pages & Pathways

Related FAQs

Why choose BioLegend recombinant proteins?

     • Each lot of product is quality-tested for bioactivity as indicated on the data sheet.
     • Greater than 95% Purity or higher, tested on every lot of product.
     • 100% Satisfaction Guarantee for quality performance, stability, and consistency.
     • Ready-to-use liquid format saves time and reduces challenges associated with reconstitution.
     • Bulk and customization available. Contact us.
     • Learn more about our Recombinant Proteins.

How does the activity of your recombinant proteins compare to competitors?

We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!

What is the specific activity or ED50 of my recombinant protein?

The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet. Please note this only applies to recombinants labeled for use in bioassays. ELISA standard recombinant proteins are not recommended for bioassay usage as they are not tested for these applications.

Have your recombinants been tested for stability?

Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.

Does specific activity of a recombinant protein vary between lots?

Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.

How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?

Use formula Specific activity (Units/mg) = 10^6/ ED50 (ng/mL)

Certificate of Analysis