How do you measure cytochrome c release?
The techniques currently used by most laboratories to measure cytochrome c release include Western blot and fluorescence micros- copy, both presenting several difficulties. The Western blot technique requires cell fractionation and isolation of mito- chondria (1,9–11).
What is cytochrome c assay?
Cytochrome c oxidase is the last enzyme in the respiratory electron transport chain of mitochondria. This colorimetric assay is based on observation of the decrease in absorbance at 550 nm of ferrocytochrome c caused by its oxidation to ferricytochrome c by cytochrome c oxidase.
Is cytochrome c a fluorescent?
The cytosolic release of Cyt c is able to dissociate the aptamer from graphene and trigger an activated fluorescence signal.
How is cytochrome c released in apoptosis?
During apoptosis, cytochrome c is released from mitochondria to the cytosol to activate a caspase cascade, which commits the cell to the death process. It has been proposed that the release of cytochrome c is caused by a swelling of the mitochondrial matrix triggered by the apoptotic stimuli.
How is cytochrome c activated?
In mammalian cells, a major caspase activation pathway is the cytochrome c-initiated pathway. In this pathway, a variety of apoptotic stimuli cause cytochrome c release from mitochondria, which in turn induces a series of biochemical reactions that result in caspase activation and subsequent cell death.
What is cytochrome c amino sequence?
Cytochrome c is a highly conserved ~12 kDa protein consisting of a single 104 amino acid peptide with a single heme group, which is covalently attached to Cys14 and Cys17. Because of its ubiquitous nature and sequence homology, cytochrome c has been used as a model protein for molecular evolution.
Does cytochrome c diffuse within the membrane?
The simplest form of this model is that upon permeabilization of the mitochondrial membrane, cytochrome c diffuses out of the intermembrane space without additional changes being necessary.
Where is cytochrome c located in the mitochondria?
In healthy cells, cytochrome c (Cyt c) is located in the mitochondrial intermembrane/intercristae spaces, where it functions as an electron shuttle in the respiratory chain and interacts with cardiolipin (CL).
How is cytochrome c bound to the inner membrane?
Cytochrome c is bound to the inner membrane by anionic phospholipids, primarily cardiolipin.
Which is necessary for the insertion of cyt c?
Indeed, CL appears to be necessary for the insertion of Cyt c into mitochondrial membranes. CL-bound Cyt c, probably does not participate in electron shuttling of the respiratory chain, but may account for the peroxidase activity recently attributed Cyt c. 2
How is PCR DNA isolated from Escherichia coli?
The PCR DNA fragment was isolated by using the QIAquick kit (Qiagen, Chatsworth, CA) and subcloned into the Nco I and Hin dIII sites of the plasmid pBAD. The plasmid was transformed into Escherichia coli and transformants were isolated by selection for ampicillin resistance.