Amount of chromatin for IgG control - URGENT! (Mar/29/2010 )
Does the amount of chromatin I use for my non-immune IgG control ChIP have to be the same as the amount I use in my actual (H3ac) ChIP? Reason I ask is, I want to try various amounts of chromatin out to check the yield and background, but if I have to match each amount with its own IgG control, I'll only be able to do half as many from my chromatin. If it isn't important I planned to use 3, 6 and 12 ug chromatin for the H3ac, and then 3 ug for the IgG. Would this be okay? Thanks.
jamessmith01 on Mar 29 2010, 08:03 AM said:
If you truly want to see how high your background is then you need to use the same amount of chromatin for both H3ac and IgG control. On the other hand, if you test your highest concentration of chromatin first and don't get any detectable background then you don't need to worry about running an IgG control for the other concentrations.
To reduce the number of concentrations you're trying I would start with an amount equivalent to 2.5 X 10^5, 5 X 10^5, and 1 X 10^6 cells, and then determine which gives you the highest ratio of H3ac to IgG. This range works for most conventional ChIP protocols. If you're doing a microplate based ChIP protocol or some other low cell number method, you should start with about 10-fold less.