I am using a ChIP protocol based on that described in Zhang L et al (2009) Amer J Nephrol 30(6):505-513, for PBMCs. The cells are resuspended in buffer and lysed by sonication, I have seen other protocols where the sample cells are lysed with buffer containing mild detergent, centrifuged at low speed to pellet the nuclei, the supernatant is removed, and the nuclei resuspended in buffer and sonicated from that point.
So my question is, is it better to isolate the nuclei from the rest of the cell contents at the beginning rather than lysing the entire cells to generate whole-cell lysate in ChIP? Or does it matter either way, if you are using an ENCODE-validated antibody? I'm just concerned that I may end up with more background if the antibody binds nonspecifically to non-chromatin proteins.
Any advice would be helpful. Thanks!
So my question is, is it better to isolate the nuclei from the rest of the cell contents at the beginning rather than lysing the entire cells to generate whole-cell lysate in ChIP? Or does it matter either way, if you are using an ENCODE-validated antibody? I'm just concerned that I may end up with more background if the antibody binds nonspecifically to non-chromatin proteins.
Any advice would be helpful. Thanks!