By Andréa C. LeBlanc
McGill Univ., Montreal, Canada. deals state-of-the-art protocols to enquire apoptosis in neurons, easy methods to become aware of key proteins fascinated with neuronal apoptosis, suggestions to check synaptosis and become aware of proapoptotic proteins in situ, and notes at the pitfalls and benefits of every approach.
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McGill Univ. , Montreal, Canada. bargains state of the art protocols to enquire apoptosis in neurons, ways to observe key proteins occupied with neuronal apoptosis, concepts to review synaptosis and observe proapoptotic proteins in situ, and notes at the pitfalls and benefits of every procedure.
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Dilute ascites fluid 1/2 with PBS and mix it with equal volume of ice-cold saturated ammonium persulfate. Stir on ice for 10 min and let it sit for an additional 10 min on ice. 3. Pellet the antibody by spinning the sample at 8000 rpm for 15 min in a Sorvall SA 600 rotor. 4. 9 (2 L each, and change the buffer solution every 4 h). 5. After dialysis, spin down the precipitated material by centrifugation at 8000 rpm for 10 min using a Sorvall SA 600 rotor. 9. Typically we use 2 mL DEAE beads per milliliter of ascites fluid.
USA 94, 11,357–11,362. 34. , et al. (1999) Bax, but not BclXL, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations. Proc. Natl. Acad. Sci. USA 96, 5492–5497. 35. , and Tsujimoto, Y. (1999) Bcl-2 family proteins regulate the release of apoptogenic cytochrome c by the mitochondrial channel VDAC. Nature 399, 483–487. 36. , et al. (1998) Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science 281, 2027–2031. 37.
The involvement of cytochrome-c in caspase activation sparked the initial interest in this area, although it has since been shown that release of SMAC/DIABLO from mitochondria can affect caspase activity by inhibition of apoptosis proteins (IAPs), causing a disinhibition of caspases (6,7). More recently, it has been suggested that apoptosis-inducing factor (AIF) is released from mitochondria and may regulate a new cell death pathway (8). Cytochrome-c plays a key role in oxidative phosphorylation by shuttling electrons between complexes III and IV of the electrontransport chain.
Apoptosis Techniques and Protocols by Andréa C. LeBlanc