problem with anti-acetyl H3 antibody - (Dec/27/2007 )

Hi everybody!

I'm trying to perform a ChIP assay using HeLa cells treated with TSA, I'm using an Anti-acetyl-Histone H3 antibody as a positive control of the experiment; I expected to obtain a poor amplification of the PCR from the immunoprecipitation with the anti-acetyl H3 Ab of HeLa cells treated with vehicle (DMSO) and an enrichment of the PCR product derived of the cells treated with TSA. However, I obtained the same level of amplications in both conditions. I'm almost sure of the treatment is working right because cell morphology changed as described before but, obviously, the positive control is wrong. Can anybody help me, please?. Thank you (Sorry for my English).

The antibody is from Upstate Anti-acetyl-Histone H3 Catalogue Number: 06-599

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Could you just be seeing background in both samples (i.e. the IP didn't work)? Did you run a mock control IP (beads only, pre-immune IgG, or peptide blocked antibody) to see what your background is like? Also, do you have primers for a negative control region (a region of the genome where you expect to see little or no acetylation in both TSA treated and untreated cells)? If you see similar amplification at this site then it's likely your IP didn't work.

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Hello, KPDE and thanks for your answer,
As you wrote, I used a IP with normal rabbit IgG and the PCR was almost clean in this case, so I think the IP is working. The problem is that the promoter region wich I'm working with is regulated by TSA, as I observed by luciferase assays with reporter constructions of this promoter region.
Thank you

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Are you performing qPCR or traditional PCR?
If traditional PCR, try decreasing the number of cycles you are performing, as maybe you have run the PCR to completion and all you are seeing is the plateau end amount of PCR product
Are you putting the same ng amount of DNA into the PCRs from both treatments?

Where are your PCR primers in relation to the genes transcriptional start site? I usually use primers directly over the TSS and about 1kb downstream, I see much more enrichment at 1kb down as the TSS undergoes histone loss.

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Hi, frozenlyse, thanks for your post,

I'm performing tradicional PCR, but I tried different conditions such as diminishing the number of cycles but the products obteined were almost the same. On the other hand, I measured the DNA quantity with a NanoDrop and, and again were practically the same. My primers amplify a 250 bp amplicon wich includes the transcriptional start site of the gene; however as I wrote previously, this promoter region is induced by treatment with TSA, so I expected a different intensity of the IP with acetyl-H3 Ab after the treatment.

Thanks.

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You should do PCR with a primer to an intergenic region (or some region where histones should not be acetylated). If you see the same PCR signal at that site as you do at the promoter then you are not seeing acetylation at the promoter. This kind of control is essential for ChIP.

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I agree with KPDE, what you need are control primers - I would think you need (at least) two sets

1. A positive control which is another genes promoter which you know definitely gets acetylated by TSA in your cell system

2. A negative control which you know should not change with TSA treatment

If these controls act as you expect, but your gene still does not I would suggest that your genes promoter is not directly regulated by TSA, but it indirectly regulated ie an upstream transcription factor of some kind is being regulated by TSA and therefore changing the activity of your genes promoter.

you could test this possibility by performing the TSA induction of gene expression of your gene in the presence of Cycloheximide, which would stop the protein synthesis of the upstream transcription factor

good luck!

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The key to your problem and almost everyone else who frequents this site
is what Frozen Lyse said; You need a positive and negative control.
Just because you treat cells with TSA doesn't mean your promoter will
be acetylated.
Therefore, go to the literature, look up a paper where people treated HeLa cells with TSA and found
the promoter to be enriched for AcH3 (NOT AcH4, they are sometimes different).
Use these primers as a positive control.
Not all genes becomes acetylated when cells are treated with TSA. Not all genes are equally acetylated
on H3 or H4.

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Thanks for all the responses!!. I'm going to perform a PCR with the oligos you wrote (positive and negative control), however I don't believe that it could be an indirect mechanism of my promoter in the response to TSA because I did a time course mRNA and I observed that this induction is so fast. Anyway I'll perform some experiments with cycloheximide.

But, my question is: Is it possible that the antibody is failing?? Is it possible that there is an increase in the H3 acetylation levels but the antibody isn't going OK??

Thank you

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Yes, your antibody could be failing but I would do the PCR 1st. Especially since you should be doing PCR with the negative control primers whether or not your PCR seems to be working.

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