6 Hour ChIP method - (Jan/07/2006 )
We've just published a method in Nucleic Acids Research (Volume 34 #1 electronic only; its free access) for doing a chromatin IP in 6 hours. This is from lysis and shearing to PCR ready DNA , though this is the fastest possible with only 4 samples or so. I usually start with already sheared chromatin with 10-16 samples and finish in about 6 hours. Not only is the assay much faster but much less labor intensive.
There are two improvements in the protocol. The first is a small improvement. We use a sonicator bath to speed up the incubation time of chromatin with antibody. Total time for precipitation (binding antibody to chromatin and antibody to prot. A beads) is 1 hour. If you don't have a sonicator bath you can still shorten the time of the assay greatly with the second improvement.
The second improvement is in the DNA extraction. We used a chelex based extraction which combines both crosslink reversal and DNA extraction into 4 simple steps that take a little over an hour (and very little labor). Not only it does take out the 6 hour crosslink reversal but eliminates phenol chloroform clean up and DNA precipitation which increase the possibility for error. The one piece of equipment this requires is a shaking heatblock (we have not tried without the shaking during heating so there's always the possibility that shaking isn't necessary).
In the paper we use quite a few cells but fewer cells still work (we just need lots of cells to get decent Cts in real time PCR for our mock IPs). We used antibodies for RNA pol II (Santa Cruz), histone H3 (abcam), hnRNP K, Sir1, and a few others and all have worked with this method. In addition we've used the method on both mammalian and yeast extracts and it has worked very well in both.
This method has dramatically increased the productivity in our lab so I encourage anyone to give it a try.
congratulations on your paper! I will certainly have look through your protocol.
I'd also like to mention, for those who've checked out the paper at NAR and would like a more detailed protocol, I have one available. Just PM me, but make sure you go to NAR and check out the paper first.
For those of you who try the protocol and either have success or difficulties, we'd like to hear about it. Also, if you try the protocol for use with ChIP on Chip or any other application other than for analysis by PCR let us know how it works. The method has been surprisingly robust in our lab (it's worked the 1st time for everyone who's tried it) but we'd like to know how it works in others hands and what modifications are necessary for use with other applications. You can contact me at:
Sorry to keep bumping this thread.