ChIP lysis buffer - Chromatin (Jul/07/2005 )
Hi,
Does anyone know which lysis buffer works best for ChIP (Chromatin Immunoprecipitation assay) I have done the literature check and come up with a lot of differences. I think the main differences are that one is SDS based and the others are not (only have Triton or NP40). Just want to know which is best, I used to do ChIP assays a year ago with SDS but now not having consistent results and want to change to a "better" protocol. Can anyone help?
Second question, does anyone have a protocol for removing cytosolic protein before Lysis? I read that this could improve final result.
Thanks
Does anyone know which lysis buffer works best for ChIP (Chromatin Immunoprecipitation assay) I have done the literature check and come up with a lot of differences. I think the main differences are that one is SDS based and the others are not (only have Triton or NP40). Just want to know which is best, I used to do ChIP assays a year ago with SDS but now not having consistent results and want to change to a "better" protocol. Can anyone help?
Second question, does anyone have a protocol for removing cytosolic protein before Lysis? I read that this could improve final result.
Thanks
Hi,
I use both. triton/NP40 are weak detergents.They're good for solubilizing cell membranes. SDS is alot stronger, so this would solubize everything. I generally use the triton for nuclear isolation and removing non-nuclear material. I use the SDS buffer for lysing the nucleus
Hope this helps.
nekko
Lysis buffer:
10ml of 1% SDS, 10mM EDTA and
50mM Tris, pH 8.1
After sonication in this buffer, make 10 times dilution with ChIP dilution buffer to make final SDS %ge to 0.1 % before addition of Ab.
Best Luck