1 reply to this topic

[emilyindallas]

Hello,

I do ChIP with the SoLiD kit for low-input (100K cells/IP) and I have had good luck validating and using antibodies against various methyl chromatin modifications. However, the IPs for anything acetyl are not going so well. I have failed to validate H2Bk12/15ac (Abcam), H4K12ac (Abcam), and H3K9/14ac (Invitrogen).

The interesting thing about the H3K9/14ac is that when I did the titration to validate it, it seemed like it worked with ~2.2 % input. However, when I did IP incubated my cells for 6 hours (+ media or + IFN), I got a flat line for the %input, with an n of 4 experiments. I recently tried another H3K9/14ac antibody from Diagenode but the best signal I got for the titration was .78% input so I am hesitant to waste experimental chromatin on this antibody.

If anyone has had any success with the low-input IP system and an antibody against an acetyl modification, please let me know!

Many thanks!

Emily

[chabraha]

I'm not really sure what the problem is...........I routinely get <1% Input values for several of my histone mods.............and its usually because I am only adding in ~.1ug of antibody................but as long as it's enriched at my region of interest when compared to my negative control region I consider the ChIP successful............for several TF's I look as as well I sometimes get %Input values ~.07%............but my negative control region is at ~0.01-0.02% so I consider the ChIP succesful