Description

Nori Porcine Caspase 3 ELISA Kit Summary

Alternative names for caspase 3: CASP3

Alternative names for porcine: pig, swine

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: 

Caspase-3 is a caspase protein that interacts with caspase-8 and caspase-9. It is encoded by the CASP3 gene and is formed from a 32 kDa zymogen that is cleaved into 17 kDa and 12 kDa subunits. The CASP3 protein is a member of the cysteine-aspartic acid protease (caspase) family.^[1] Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. Caspases exist as inactive proenzymes that undergo proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme. This protein cleaves and activates caspases 6 and 7; and the protein itself is processed and activated by caspases 8, 9, and 10. It is the predominant caspase involved in the cleavage of amyloid-beta 4A precursor protein, which is associated with neuronal death in Alzheimer’s disease.^[2]  Caspase-3 is activated in the apoptotic cell both by extrinsic (death ligand) and intrinsic (mitochondrial) pathways.^[3] Caspase-3 functions to inhibit XIAP activity by cleaving caspase-9 at a specific site, preventing XIAP from being able to bind to inhibit caspase-9 activity.^[4] Caspase-3 has a typical role in apoptosis, where it is responsible for chromatin condensation and DNA fragmentation.^[5] Elevated levels of a fragment of Caspase-3, p17, in the bloodstream is a sign of a recent myocardial infarction.^[6] Caspase-3 may play a role in embryonic and hematopoietic stem cell differentiation. Caspase-7, apoptosis-related cysteine peptidase, also known as CASP7, is a human protein encoded by the CASP7 gene. Caspase-7 is a member of the caspase (cysteine aspartate protease) family, and has been shown to be an executioner protein of apoptosis. The precursor of this caspase is cleaved by caspase 3, caspase 10, and caspase 9. It is activated upon cell death stimuli and induces apoptosis. Caspase 7 has been shown to interact with Caspase 8,^[7] Survivin^[8] and XIAP.^[9]

References

1. Alnemri ES, et al. (1996). “Human ICE/CED-3 protease nomenclature”. Cell. 87 (2): 171.
2. Gervais FG, et al. (1999). Cell. 97 (3): 395–406.
3. Salvesen GS (2002). Cell Death and Differentiation. 9 (1): 3–5.
4. Denault JB, et al. (2007). The Biochemical Journal. 405 (1): 11–9.
5. Porter AG, Jänicke RU (1999). Cell Death and Differentiation. 6 (2): 99–104.
6. Agosto M, et al. (2011). Journal of the American College of Cardiology. 57 (2): 220–1.
7. Srinivasula SM, et al. (1996). Proc. Natl. Acad. Sci. U.S.A. 93 (25): 14486–91.
8. Shin S, et al. (2001). Biochemistry. 40 (4): 1117–23.
9. Deveraux QL, et al. (1997). Nature. 388 (6639): 300–4.

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