I'm trying to western blotting caspase3 from in vitro culture protein extracts but after different times under a same condition. In the higher times of treatments I can see lesser amount of protein immunodetected in loading control and caspase3 because cells starts to die (as expected) and suspend in the media which is discarded before doing the extraction, so there will be differences between immunodetecting proteins from the control and the treated culture due the amount of cells that remain adherents. So... I will start to do Bradford assay before loading the extracts, but any additional advice?
Cynthia- yes, definitely measure protein concentration before loading. It is very important and you can use bradford (quick but less sensitive), BCA (longer, but more sensitive and is more compatible with different lysis buffers) or other techniques. You can also do a Western blot for a loading control protein (like tubulin). However you should ask yourself, will you get any useful information from doing a Western blot on extracts of dead (or very close to dead) cells? So many things happen at that stage that whatever you see might have nothing to do with your topic of interest.
You may want to consider measuring caspase 3 activity. There are many commercial substrate available for this.
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