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why is my histone h3 more in the cytosol than the nucleus?


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#1 tfcheng

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Posted 04 May 2010 - 07:54 AM

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

#2 bob1

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Posted 05 May 2010 - 04:20 PM

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.

#3 huotoad

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Posted 20 May 2010 - 08:46 PM

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?

#4 bob1

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Posted 23 May 2010 - 06:04 PM

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




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