![]() The described methods can be applied to any particular protein of interest.Īpoptosis Caspases Cleavage Extrinsic Pathway Proteins Western Blot. Western blot is a powerful technique widely used in scientific and medical research to separate and detect various key proteins, especially the less abundant ones in critical diagnostic samples. We detail protocols for protein extraction, quantitation, casting, and running gel electrophoresis and western blot analysis of caspase -8 and caspase -9 activation. In this chapter, we describe the step-by-step methodology in the western blot analysis of caspase cleavage during apoptosis. In the context of apoptosis, the proper analysis of western blot results depends on the understanding of the mechanisms and outcomes of caspase processing during the course of its activation. Detection of caspase cleavage by western blot analysis is a conventional method to demonstrate the induction of apoptosis. Electrophoresis used to separate proteins according to their electrophoretic. Transfer to a solid support (Blotting) Marking target protein using a proper primary and secondary antibody to visualize (Detection). ![]() Active caspases cleave cellular substrates and are thus the main effectors of the apoptotic cell death pathway. The technique consists of three major processes: Separation of proteins by size (Electrophoresis). Caspases are synthesized as inactive pro-caspases and activated by a series of cleavage reactions. As an analytical biochemistry assay and a 'wet lab' technique, ELISA involves detection of an analyte (i.e., the specific substance whose presence is being quantitatively or qualitatively analyzed) in a liquid sample by a method that continues to use liquid reagents during the analysis (i.e. Apoptosis is executed by a class of cysteine proteases called caspases. Apoptosis is a type of programmed cell death induced by a cascade of biochemical events, which leads to distinct morphological changes characterized by cell shrinkage, membrane blebbing, chromatin condensation, and DNA fragmentation. ![]()
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