The Human Caspase 3 (Cleaved) ELISA Kit is a solid-phase sandwich Enzyme-Linked Immunosorbent Assay (ELISA) designed to detect and quantify the level of Caspase 3 (Cleaved) in fresh or frozen human cell lysates. The assay recognizes both natural and recombinant Caspase 3 (Cleaved).
Principle of the method
A monoclonal capture antibody specific for Caspase 3 (Cleaved) has been coated onto the wells of the 96-well plate. During the first incubation, standards of known content and unknown samples are pipetted into the wells and the antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for the target protein is added to the wells and serves as a detection antibody by binding to the immobilized protein captured during the first incubation. After washing, a horseradish peroxidase labeled anti-rabbit IgG is added. This binds to the detection antibody to complete the four member sandwich. After a third incubation and washing to remove all the unbound enzyme, a substrate solution (TMB) is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of target protein present in the original specimen and the optical density can be read on a standard microplate reader.
Each manufactured lot of this ELISA kit is quality tested for criteria such as sensitivity, specificity, precision, and lot-to-lot consistency. See manual for more information on validation.
Caspases are a family of aspartate-specific cysteine proteases that act in a step-wise signaling manner like kinases. Caspases are present in all cells, recruitment of these proteases to oligomerized receptors leads to activation accompanied by autoproteolytic cleavage. Active caspases can proteolyze additional caspases generating a caspase cascade that cleaves proteins critical for cell survival. The final outcome of this signaling pathway is a form of controlled cell death termed apoptosis. The subgroup of caspases involved in apoptosis is called initiators or effectors. Caspase-3 cleaves substrate at the carboxyl terminus of aspartate residues. Active caspase-3 has two active sites and consists of two identical large (~ 20 kDa) and two identical small (~ 10 kDa) subunits that are derived from two precursor caspase-3 polypeptides. Caspase-3 is proteolytically activated by other caspases. Both subunits contribute to substrate binding and catalysis. The active site cysteine that covalently binds the substrate is located near the C-terminus of the large subunit. Active caspase-3 has two-fold symmetry, two active site pockets each residing on an opposite side. Caspase-3, together with caspases 8 and 9, is situated at pivotal junctions in apoptotic pathways. Caspase-3 appears to amplify caspase 8 and 9 initiation signals into complete commitment to apoptotic disassembly.
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