3 replies to this topic

[tfcheng]

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

[bob1]

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.

[huotoad]

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?

[bob1]

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