Titrate chromatin for chIP - (Jan/15/2007 )

Hi everybody!

I would want to know how do you titrate your chromatin before doing a chIP experiment?

Thanks in advance.

If you are using cells from TC, I would suggest that you measure the number of cells you would typcially get in a prep. Then figure what percentage of your prep is equivalent to 1 to 2 million cells. A ballpark figure that many use is about 1 to 2 million cells per ChIP using 1 to 2 ug of antibody (the amount of antibody is dependent on how much your protein decorates the DNA).

The ideal situation is to have enough chromatin to just saturate the binding capacity of your antibody and nothing more. Any additional chromatin will just add to your background. If you have chromatin already prepared and don't have any idea how many cells it is equivalent to you might try a few dilutions of your chromatin on ChIP and see which gives you the best ratio of signal (ChIP with your antibody of inerest) to background (ChIP with pre-immune IgG or just protein A beads without any antibody). Also keep in mind that, if you are using PCR to quantitatively or semiquantitatively analyze your results, you need to use enough chromatin so that the PCR cycles are within the linear range of your primer.

Hope that helps.
Joel

Thank you very much for your help!