Cytochrome c as well as caspase 3 (or other caspases)? - (Nov/28/2006 )
If you already checked for caspase activity (caspases 2 and 3) to see if a cell was undergoing apoptosis, would you bother to double check a western blot for cytochrome c to double check that it is cytochrome c that is activating the caspases (though the apoptosome)?... or is that a waste of time (precious time) if you already had caspase activity data?
I'm curious to what you all think as I'm not sure if I want to do that or if it's overkill. Any opinions or experience?
Thanks for any advice.
I would say that it depends on the importance of the assay. if it is a main pillar of whatever you want to prove, I would double-check. even though caspase assays are quite credible. But I would do a PARP Western blot since I've had lots of trouble doing Cyt c Western Blots :-)
Caspase 3 is at the end of caspase cascade. So I think that it is enough for apoptosis activation. Moreover you can look for DNA laddering to double check apoptosis. DNA fragmentation in fact is the final step of apoptosis so with this assay you're sure that apoptosis is activated.
Yes I was also going to do a TUNEL assay for DNA laddering. Is a cytochrome c blot a good indicator of apoptosis....my only hesitation would be wondering if the cell lysis also disrupts mitochondrial outer membrane which would release cyt c anyway! Wouldn't all cells look similar then? Does lysis by passage through a needle lyse mitochondria (release contents, including cyt c)? Then I would have to use an alternate lysis method.