Description
Principle of the Assay::The Human Cytochrome c ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Human Cytochrome c in Cell Lysates. This assay employs an antibody specific for Human Cytochrome c coated on a 96-well plate. Standards and samples are pipetted into the wells and Cytochrome c present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-Human Cytochrome c antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of Cytochrome c bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
Background/Introduction: Human somatic cytochrome c is a 15 kDa, 104 amino acid polypeptide that participates in both oxidative phosphorylation and apoptosis. It exhibits 91% aa identity with mouse/rat cytochrome c. The molecule is initially synthesized in the cytoplasm as an extended, 12 kDa apoprotein. This molecule is subsequently transported across the outer mitochondrial membrane into the intermitochondrial space via a receptor-independent process. Here, it associates with an inner mitochondrial membrane enzyme called CCHL which covalently attaches one heme molecule to the N-terminus, forming functional cytochrome c. This covalent attachment causes cytochrome c to undergo a conformational change to a globular molecule effectively trapping cytochrome c in the intermitochrondrial space. Within the intermitochondrial space, cytochrome c is actively involved in the oxidative phosphorylation pathway. It transports electrons from the cytochrome c reductase complex to the cytochrome c oxidase complex. This transports excess electrons along the respiratory pathway and generates ATP for energy-dependent processes