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why is my histone h3 more in the cytosol than the nucleus? - (May/04/2010 )

Hi,

I am doing cyto/nuc fractionation, when I use Histone h3 (Santa Cruz, sc-10809) as nuclear marker, I always found it's more in the cytosol than the nucleus. I use low salt/high salt protocol to get my fractions, and besides H3 I also probe PARP1, which is mostly in the nucleus, what gives? thanks!

TFC

-tfcheng-

Low salt/high salt fractionations are not perfect, you will get some cytoplasm in the nuclear fraction and some nuclei in the cytoplasm. If you want a clean prep you need to do a centrifuge gradient method.

-bob1-

bob1 on May 5 2010, 05:20 PM said:

Low salt/high salt fractionations are not perfect, you will get some cytoplasm in the nuclear fraction and some nuclei in the cytoplasm. If you want a clean prep you need to do a centrifuge gradient method.



do you mean that there are lots of cytoplasm protein in the nuclear extract if I am using a low /high salt method?

-huotoad-

Unless you are doing a centrifuge gradient/cushion protocol, your preps will not be clean.

-bob1-