How to reduce the viscosity of Nuclear extractions??

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Mon Feb 7 09:02:31 EST 2000


"G. Dellaire, Ph.D." wrote:

> Hello,
>
> Does anyone out there have a good method for reducing the viscosity of
> nuclear extracts (mammalian) after high salt extraction?  I am having
> trouble loading these extracts on SDS-PAGE gels as they are so bloody
> viscous.  I have found that using a sonicator (barrel/tip) is not
> practical if you have dozens of samples or if you have very small
> volumes (under 100 ul to 30 uls typically).  The resulting foam eats up
> most of your sample.
>
> I have heard about using a sonicating water bath, has anyone used one of
> these for this use?
>
> any and all tips/replies are welcome.
>
> Cheers,
>
> Graham Dellaire
>

Hi Graham,
The high viscosity results from DNA that is released from nuclei upon
treatment with high salt. There are several options to reduce or eliminate
viscosity. First of all, ultracentrifugation of the samples is a good way
to pellet most of the DNA (30min, 150.000g or more) and reduce viscosity.
In addition, you can remove lipids that float on top of the sample after
centrifugation. We´ve been using for year ultracentrifugation for making 1M
NaCl nuclear extracts as starting material for protein purification, and it
never failed.
A second option is to treat the extracts with some nuclease. This is also
quite gentle, but you´ll have to check which nuclease does the job.
Micrococcal nuclease is a good start, but most commercial preparations are
contaminated with low amounts of proteases, and might eat up your
protein....
A third option, if you only want to analyse the samples by SDS PAGE and
don´t need native protein, is to separate DNA and proteins by a
chloroform/methanol precipitation. This method does not really reduce the
viscosity of the sample at first (better do that with sonication), but
gives great western blots from total cell samples because you essentially
"purify" proteins away from lipids and nucleic acids.
I do it this way: to your extract (say 100ul), add SDS to 1% final
concentration, shake well to mix, and if possible sonicate for 20" , then
add 4 volumes of methanol (400ul).  Vortex again, then  add 1 volume
(100ul) of chloroform, vortex vigorously, then spin for 2 min. Remove the
upper phase containing DNA, and add 4 volumes of MetOH (400ul) to the
remaining interphase/organics phases. Vortex again to mix well, then spin
out the proteins for 5min. Air-dry pellet and redissolve in SDS PAGE sample
buffer.

hope this helps,
Frank





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