Nori Rat Serpin A4 ELISA Kit Summary

Alternative names for Serpin A4: SERPINA4, Serpin Family A Member 4, kallistatin, Kallikrein inhibitor, Peptidase inhibitor 4, PI-4, KST, PI4

Q5M8C3

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Serpin A4 (Serpin Family A Member 4) is encoded by SERPINA4 gene and is a member of serpin family. An important paralog of this gene is SERPINA11. Among its related pathways are platelet activation, signaling and aggregation. It inhibits human amidolytic and kininogenase activities of tissue kallikrein. Inhibition is achieved by formation of an equimolar, heat- and SDS-stable complex between the inhibitor and the enzyme, and generation of a small C-terminal fragment of the inhibitor due to cleavage at the reactive site by tissue kallikrein. Serpins are a superfamily of proteins with similar structures that were first identified for their protease inhibition activity and are found in all kingdoms of life.^[1] The acronym serpin was originally coined because the first serpins to be identified act on chymotrypsin-like serine proteases (serine protease inhibitors).^[2] They are notable for their unusual mechanism of action, in which they irreversibly inhibit their target protease by undergoing a large conformational change to disrupt its active site.^[3] This contrasts with the more common competitive mechanism for protease inhibitors that bind to and block access to the protease active site.^[5][6] Protease inhibition by serpins controls an array of biological processes, including coagulation and inflammation, and consequently these proteins are the target of medical research.^[4] Their unique conformational change also makes them of interest to the structural biology and protein folding research communities.^[3] The conformational-change mechanism confers certain advantages, but it also has drawbacks: serpins are vulnerable to mutations that can result in serpinopathies such as protein misfolding and the formation of inactive long-chain polymers. ^[5] Serpin polymerisation not only reduces the amount of active inhibitor, but also leads to accumulation of the polymers, causing cell death and organ failure.^[4] Although most serpins control proteolytic cascades, some proteins with a serpin structure are not enzyme inhibitors, but instead perform diverse functions such as storage.

References

1. Silverman GA,et al. (2001) The Journal of Biological Chemistry. 276(36): 33293–6.
2. Silverman GA,et al. (2010) The Journal of Biological Chemistry. 285(32): 24299–305.
3. Whisstock JC, et al. (2006)  Current Opinion in Structural Biology. 16(6): 761–8.
4. Stein PE, Carrell RW (1995) Nature Structural Biology. 2(2): 96–113.
5. Janciauskiene SM, et al. (2011). Respiratory Medicine. 105(8): 1129–39.