Like most people, I'm using the upstate kit to perform my first ChIPs, and like most people I'm running into problems. Don't even get me started on Upstate's fast-talking about how they give you enough reagents for 22 IP's but only enough for 10 lysis/sonication events.
I'm using LNCaP cells and searching for nuances in p53 acetylation at the p21 locus upon HDAC inhibitor treatment. LNCaP cells are very very picky, and they do not like to be too crowded (or too sparse for that matter) in the culture dish. I can fit 10e6 cells in a 10cm dish, possibly 2X10e6 if I'm pushing it. Scaling up, I can get 4.5X10e6 cells into a 15cm dish, but that's REALLY pushing the limits of these cells. If I'm talking 96 hour timepoints, I can't put any more than 10e6 into the plate or they overgrow and start dying - as you can imagine, this kind of screws with the p53 status of the cell.
But this relates to my problem: If I'm using 10e6 cells/dish/sonication, I don't have any capacity to dilute the SDS lysis buffers in ChIP dilution buffer. This means I'm heading into the IP steps at a full SDS concentration. Won't this interfere with the antibody binding?
The protocol they supply is built around 10e7 HeLa cells and they only give passing remarks on what to do in other cases, with none of those remarks actually providing any solutions.
Has anybody successfully and reliably performed ChIP on a sample without usng the ChIP dilution buffer? Does anyone have any suggestions here?
Edited by MunkySpunk, 28 July 2009 - 06:02 AM.