ChIP cell lysis buffer - what lysis buffer do you use for Chromatin IPs (Jan/29/2008 )

Hi everybody,

I got some questions on cell lysis after x-linking the cells...
What lysis buffer should one use? There are lots of different protocolls were people use different lysis buffers...
On the one hand people lyse their cells directly by e.g. SDS-Lysis buffer (1%) on the other hand there are protocols were people first lyse the cells and isolate the nuclei and afterwards lyse the nuclei...? Are there any advantages?

What are the differences in using RIPA or SDS-lysis buffer?
Or is it depending on the cell line? I am working with transfected HEK 293 cells...

Thank you for your help...

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With SDS buffers, some people get better shearing results than with non-ionic detergents. You just need to make sure that the SDS is diluted out (to 0.1% or less) and replaced with non-ionic detergents (tritonX, NP-40) before you try to run your IP. On the other hand we use a buffer with triton X and NP-40 (IGEPAL) and the sonication efficiency is just fine and we don't have to worry about SDS mucking up the IP.

The advantage to giving the nuclei a wash or two before sonication (as you put it, "lysing the cells before lysing the nuclei") is that you remove most of the soluble proteins. When doing ChIP you only want to pull down your protein of interest if it is bound to the chromatin (insoluble before sonication) rather than any of the unbound protein (soluble). The unbound protein will compete with the bound protein for your antibody and decrease your IP efficiency.

Also, keep in mind, when referring to "nuclei" from cells that have been fixed with formaldehyde and then lysed, what's really being referred to is a complex made up of the nuclear matrix, chromatin, and a portion of the cytoskeleton, since this has all been crosslinked together.

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Thank you very much for your help...

Just one further question: how much buffer do you use for cell lysis? e.g. Normally I use 2-5 x 10e7 cells and I use 1 ml SDS lysis buffer...
Is this ok?

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That's pretty close to what I use but I know that I use too many cells for the amount of buffer. I often get lipid floating at the top of my sonicated chromatin which, if I used more buffer, would get solublized. I've never worried about this though and my ChIPs work fine (as far as I can tell).

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Hi, can i just check with you guys if Triton X, prior to sonication in water bath with SDS lysis buffer, is able to give a cleaner background since it extracts the nucleus and eliminates non-nuclear material, thus preventing the non-nuclear protein from competing with the nuclear protein? coz Im testing on nuclear receptor. what im afraid is that the non-ligand-bound nuclear receptor (still in cytoplasm) may compete with the ligand-bound nuclear receptor (translocated into nucleus) for Antibody during immunoprecipitation if I just do a water-bath sonication in 1% SDS lysis buffer.

I tried both methods, with and without Triton, but I did not get consistent results so it's hard to compare. Logically speaking, triton x treatment should give 'cleaner' RT- signal but my non-ligand treatment sample had really high % input. Even when I did RT-pcr using primers for non-specific genomic regions, the % input was relatively high too (>0.1% input) when usually i get 0 or 0.01% for the random regions.

Can anyone help me on this? Any similar encounters or those who have tried both methods before?

Thanks alot in advance..

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We lyse the cells with non-ionic detergent buffer (plus one wash in the same) prior to sonication. This gets rid of most of the soluble proteins but will not remove insoluble proteins (e.g. cytoskeletal proteins or those associated with the cytoskeleton) since these are crosslinked in a complex with the nucleus and some remnants of the ER and golgi.

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I think it depends very much your cell type, protein you are looking at and antibodies.

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