How much cytoplasmic and nuclear extracts load in a WB? - (Sep/20/2011 )
I'm trying to assess protein expression in nuclear and cytoplasmic fractions. I was recommended to load three times more total protein of cytoplasmic than nuclear extracts. This was because nuclear proteins are more concentrated in the nuclear fraction. In literature I've seen that is more frecuent to load equal amounts of both extracts. In your opinion what is more appropiate?
I agree on loading a lot more of cytoplasmic fraction than nuclear extract. I have seen that when I load same amount of protein between nuclear and cytoplasmic extract, I get a much more faint band as opposed to nuclear extracts.
depends on the protein. if working on something like Actin, with the lowest amount of cytoplasm fraction you can see it. I normally load 30 ug on BioRad-mini. But I have gone p to 60 ug although it gives smear on the SDS-PAGE.