Hello,
I'm recently trying to focus on H3K27ac histone modification for one of my research.
Unfortunately, I could not perform any successful experiment, and try to troubleshoot my experiment as following:
1) Sonication: I've optimized sonication as first step; also controlled my input control's by running agarose gel, and DNA is pretty well fragmented.
2) IP conditions: I've optimized the antibody amount, also optimized the salt concentration.
3) Cross-linking & Reverse Cross-linking: Previously (for same cell line) optimized amounts are used.
Are there any other leads that I could follow? Could it be about acetylation detection, i.e. no usage of deacetylase inhibitors?
Thanks for all the help...
Hi Ali,
Have you verified whether the H3K27ac modification is present, and at what amount, in your particular sample using an ELISA-based quantification method or other technique? Also, is the antibody validated for use in ChIP?
Hi Ali,
In addition to Michael's comments, I have some other suggestions.
What is the size range of your sonicated fragments? Sometimes, using MNase digestion gives more reproducible nucleosomal fragments that are more readily immunoprecipitated with a histone antibody, so you may want to consider that.
Do you already add sodium butyrate or a similar HDAC inhibitor to your lysis and sonication buffers? A good starting range is 2-5mM sodium butyrate.
Thanks,
Barry
Thanks for the answers Michael Spelios and Barry Zee ,
Michael, I've verified H3K27ac modification involvment. The antibody is Diagenode H3K27ac ChIP grade antibody.
Barry, the sonicated fragments are 100-500 bp. Adding HDAC inhibitor does solved the case, thanks.
Now my concern is that using HDAC inhibitor could lead to false increase or decrease results, i.e. Normally control cell's could have 1Unit H3K27ac, while treatments cells have 5Unit, the difference would be 5 folds. Whereas when we add HDAC inhibitor, the acetylation could accumulate and Control cells could have 5 Unit H3K27ac, while treatment would increase to 10 Unit, and the difference would be observed as 2 fold. Or totally opposite could happen.
Do you have any suggestions for this case?
Thanks,
Cenk
Hi Cenk,
It's an interesting question about the "false" induction of hyperacetylation due to HDAC inhibitors (HDACi). Generally though, HATs or histone acetylases are not very active once you lyse your cells because the acetyl-coA co-factor is greatly diluted in lysis buffer. Since you presumably add the lysis buffer+HDACi on ice as well, one would predict ectopic HAT activity to be low.
In this paper attached (Article Quantitative Dynamics of the Link Between Cellular Metabolis...
), generally histone acetylation has a "turnover" of 40-60 minutes. Assuming H3K27ac turnover has a similar turnover in your cell type and conditions, one would predict that as long as you keep your lysis conditions relatively short with HDACi, any ectopic HAT activity should not significantly increase acetylation levels. It certainly should not double the acetylation levels in control if your lysis duration is 5-10 minutes on ice.If in fact you are seeing a difference from 1 Unit to 5 Units, it's possible that the former calculation (i.e. 1 unit) is actually an underestimation of the true acetylation level in your control. You could also titrate the HDACi concentration to slightly lower level (maybe 2 fold). Usually the inhibitors are used at vast excess over what's needed.
I hope my comments above make sense.
Thanks,
Barry
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