homgenize and lyse the tissue together with sonication - feasible ? Why not? any comments? (Jun/29/2005 )

hi,

Since the sonicator can homgenize the tissue ,
could I homgenize and lyse the tissue together
with sonication ? feasible ?
Why not?

with running gel,I checked the sonicated lysate ,
which was homgenize and lyse the tissue together
with sonication , I got the sonicated DNAs centering ca.500bp
well.

However , the standard ChIP protocol is step-by-step,
i.e. cut the tissue in small pieces, isolate the nuclei,
SDS lysis ,etc, then sonication.

When sonicator homogenizes the tissue piece, and the
lysis process as well , and I always add sufficient
Roche Complete Mini inhibitor cocktail . My procedure
will causes higher background ? has anybody done as me ?

any comment s most appreciated in advance!

ZY

Hi ZY,

I have not done Chip in the manner you have described or heard of others doing so also.

Chromatin exists in the nucleus and it is more ideal to isolate nuclei and shear the isolate. If you take whole cell lysates, sure you have protease inhibitor cocktails, but I would say you also increase the number of targets for your antibody because there are some many more proteins from the cytoplasm and the cellular membranes. I would say you will get a high background with very little yield of actual chromatin.

Again I have not heard of people doing it this way and I would be interested to hear how you went.

Good luck!

Nick

hi, Nick,

one friend mentioned he has done so suceessfully . i'm trying it.
it's logical to have more bk; but I cannot figure out too much reasons that
it will affect the products of actual chromatin as you suggested.
why less? if fixation ok, ...

thank u & look forward to ur new reply in forum.

ZY

Hi ZY,

I would say that the cytoplasmic fraction of proteins aren't a part of chromatin and thus you will be spending your Ab on binding of free protein and not chromatin associated protien therefore you would essentially get less IP.

Having said this I suppose you can account for this by adding more Ab to your IP.

That was my reasoning behind it all. Again this reason will fall to bits if the protein is not abundantly translated in the cell.

Nick