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Brief Description & Uses The permeabilization of mitochondrial outer membrane and the subsequent release of cytochrome c and other apoptogenic proteins from mitochondrial intermembrane space into the cytoplasm is considered a hallmark of many apoptotic pathways. Therefore assaying these proteins in mitochondrial and cytoplasmic fractions is a prime interest of many researchers. Most of the current biochemical methods of quantification of cytoplasmic and mitochondrial cytochrome c involve mechanical disruption of cells to obtain mitochondria-enriched and cytosolic fractions. These methods are time-consuming and limited to a small number of samples, and they also include the risk of disrupting the mitochondrial outer membrane, which can cause the release of cytochrome c in non-apoptotic cells. Kit MSA11 allows researchers to induce apoptosis in cultured cells using their preferred method, and then to rapidly and gently isolate a cytoplasmic fraction and a cytoplasm-depleted cell pellet containing intact mitochondria using a proprietary detergent and protocol. This kit avoids the need to resort to time-consuming and inefficient cell disruption and differential centrifugation. The two isolated fractions can then be analyzed using the included cocktail for Western blotting that includes antibodies against cytochrome c, a cytoplasmic marker protein, and two mitochondrial marker proteins. These antibodies provide an effective means to ensure that the experiment has been appropriately controlled and that valid fractions have been obtained. The antibody cocktail is also available for purchase separately: MSA12 ApoTrack™ Cytochrome c Apoptosis Cocktail. Specifications
Cocktail Components
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Images MSA11 includes an optimized cocktail of 4 monoclonal antibodies for Western blotting. The cocktail includes an antibody to determine if cytochrome c has been released from the mitochondria (M) into the cytoplasm (C) fraction triggering apoptosis. Also included are a cytoplasmic marker (GAPDH), a mitochondrial matrix marker (PDH E1 alpha) and a mitochondrial inner membrane marker (ATP synthase F1 alpha). Note that apoptosis has been induced in this figure by two mechanisms staurosporine (STS) and FAS antibody (FAS). Also note that different cell types respond differently to these treatments, compare Jurkat vs. HeLa vs Osteosarcoma 143B cells.
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