ChIP crosslinking results in precipitate!?
Posted 16 November 2010 - 11:07 AM
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?
Posted 19 November 2010 - 03:48 AM
Posted 23 November 2010 - 12:21 PM
Posted 24 November 2010 - 08:13 AM
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...
You may also want to take a look at this:
Biotechniques. 2005 Nov;39(5):715-25.
Two-step cross-linking method for identification of NF-kappaB gene network by chromatin immunoprecipitation.
Nowak DE, Tian B, Brasier AR.
Posted 26 January 2011 - 02:30 PM
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...