4 replies to this topic

[laichiehmin]

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

[ezilybored]

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.

[laichiehmin]

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...

[KPDE]

laichiehmin, on 23 November 2010 - 12:21 PM, said:

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.

[Radish]

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...