ChIP crosslinking results in precipitate!? - (Nov/16/2010 )

Hi all, has anyone else observed precipitate in the initial crosslinking step of ChIP? If so does it significantly affect analysis later on?

I start with ~1x10^7 cells (tried Jurkat and U2OS, saw same thing in both), resuspended in 10ml cold PBS, added formaldehyde to 1% and left at room temperature with rotation for 20 min. Afterwards I saw precipitates.
I quench with glycine (0.125M glycine) for 5 min. at room temp and precipitate persists.
When I tried to spin this down at 2000g for 5 min., it would not pellet. I had to do max speed on tabletop for 1 min.
I proceed with the rest of the ChIP protocol, and have to use max speed/1min spins for PBS wash, cell lysis and nuclei lysis.

I've noticed that my rough chromatin yield is very low. In addition, even though I have a very large cell pellet at the beginning of the protocol, after crosslinking, the pellet is barely visible. It would seem I'm losing a lot of cells? Or maybe my cells are already lysing in the formaldehyde?

Any thoughts or suggestions?
Thanks

Are the cells fully resuspended when you fix them? They may just become more visible when fixed. I worked with dictyostelium before and they become more visible when fixed. I have no explanation for why but they did. Also, are you sure you need to fix for 20minutes? That does seem longer than most people fix for.

I try to fully resuspend before fixing. The solution is definitely not totally clear due to the cells inside. But there is definitely no visible clumps of cells. Maybe I will try using a bigger volume to dilute the cells out more. Also, I'm trying 20min fix because I'm trying to chip a transcription factor and at 10min not getting a good signal. Maybe I will try 15min...

laichiehmin on Tue Nov 23 20:21:21 2010 said:

Hi KPDE

That paper changed my life lol

I spent a year trying to get ChIP to work, and it only took 3 weeks after that paper...