Cross-linking and cross-link reversal for ChIP - (Mar/07/2006 )
Right - a few months ago I started 'ChIPing' using the Upstate kit. I have optomised the sonication (thanks to helpful suggestions from this site regarding time and volume) and I have DNA in my input controls. However, there is an ominous lack of DNA in my 'ChIPed' samples. If any of you can answer these questions I'd be REALLY grateful,
(i) is the cross-linking with formadehyde very time or temperature sensitive? At the moment I follow the protocol, 1% formaldehyde (molecular grade) in total 1ml volume at 37C for 10 minutes. I add the formaldehyde at 2x concentration in 500ul to 500ul of cell suspension so that it mixes efficiently and quickly (one of you suggested that and it works very well, thanks!). As I get no product at the end of the protocol I wonder if the cross-linking is not good enough?
(ii) is the reversal of cross-linking with NaCl very time or temperature sensitive? Again, following protocol, 65C for 4hrs (what a pain!!) with NaCl
(iii) protease inhibitors - I don't trust mine! They are clearly very important as I need intake histones for the immunoprecipitation. I follow the protocol with final conc 1mM PMSF, 1ug/ml aprotinin and 1ug/mlpepstatin A). Please can someone tell me what they use and where the get it from??!!
Thank you - I really want this to work as it will make my PhD!!!
I suspect your problem is with your antibody. Are you sure that your antibody is working well under fix conditions? Many antibodies will work perfectly for normal IP but not for ChIP because of the fixation step. I don't think the reverse cross-link is your problem since the DNA in your "input control" is OK. As for the protease inhibitor, I suggests the Roche Complete Protease inhibitor Cocktail Tablet.
Are you working with suspension culture or adherent culture? If it is adherent culture, according to the protocol formaldehyde should be added to the cells before they are harvested.
For reverse crosslinking, 4 hrs is a minimum and can be longer.
we reverse crosslinks overnight!