How to test ChIP cross-linking and reversal? - (Jun/13/2006 )
Dear KPDE
Thanks a lot
Danilo
Dear KPDE
I would also like to try your new protocol for ChIP. Is it possibile for you to send me the protocol or a reprint of the paper in nature protocols as we do not have access to this journal?
Thanks in advance
Grooya
I was wondering how well/if this fast ChIP method could be adjusted to using spin columns (instead of pipetting off the supernatant, just spinning.
You mean, just loading the beads and everything into a spin column and spinning out the supe? I vaguely remember hearing about someone doing this and being successful but unfortunately I can't remember where. Are you worried about carry over of beads during sample transfer, the large amount of dead volume, or just trying to speed up the assay?
Actually, I want to scale it down such that the 'pellet' may be too small to distinguish. Plus my supernatant recovery skills aren't great (seems like too much variability). I just tried it, so we'll see what the PCRs look like...
EDIT: Well, no luck. But not the no luck I was thinking would happen. The antibody (two different abs), no antibody, and IgG samples all show PCR signal 
. What the heck? Any help here. At least the first time there was more signal in the two antibody samples than the no ab sample, now they are all roughly the same. I even tried washing with a higher (450mM) salt concentration in the buffer this time. ANY HELP OUT THERE?
You mean, just loading the beads and everything into a spin column and spinning out the supe? I vaguely remember hearing about someone doing this and being successful but unfortunately I can't remember where. Are you worried about carry over of beads during sample transfer, the large amount of dead volume, or just trying to speed up the assay?
EDIT: Well, no luck. But not the no luck I was thinking would happen. The antibody (two different abs), no antibody, and IgG samples all show PCR signal
. What the heck? Any help here. At least the first time there was more signal in the two antibody samples than the no ab sample, now they are all roughly the same. I even tried washing with a higher (450mM) salt concentration in the buffer this time. ANY HELP OUT THERE?You mean, just loading the beads and everything into a spin column and spinning out the supe? I vaguely remember hearing about someone doing this and being successful but unfortunately I can't remember where. Are you worried about carry over of beads during sample transfer, the large amount of dead volume, or just trying to speed up the assay?
After the antibody incubation with the chromatin, did you centrifuge (10min top speed, at least) and be careful to take only the top 85-90% (or less) when transferring your chromatin to the protein A beads?
Well, I did it through a column, so I spun it and collected the flowthrough. I am about to do it again without the columns, but this time preclearing my samples with some beads. Maybe that will get rid of the pcr signal in the no ab samples! I also saw suggested blocking the beads with BSA. Which do you think would work better (or both)?
You can try incubating your beads with 5% BSA and 100ug/ml sheared salmon sperm DNA prior to and during the incubation with antibody and chromatin. This may help reducing the non-specific binding. Also, if you are using high capacity beads (like the Amersham Fast-Flow protein A agarose) you can reduce the amount of beads you use.
I would reduce the amount of beads, but as it is the major (new) problem I'm having is disappearing agarose beads? I spin tubes that just had a pellet (all I did was add my incubated ab-sample) but after spinning I no longer see a pellet Any thoughts?
You can try incubating your beads with 5% BSA and 100ug/ml sheared salmon sperm DNA prior to and during the incubation with antibody and chromatin. This may help reducing the non-specific binding. Also, if you are using high capacity beads (like the Amersham Fast-Flow protein A agarose) you can reduce the amount of beads you use.