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Acetylated Histone 4 - visualization (Aug/31/2005 )

Hey there,

I am doing western blot for acetylated histones 3 and 4. H3 works well, but I cannot get a signal for H4. Have tried 2 different Abs and several concentrations.. nothing. I even loaded 40ug protein, still nada!

Any suggestions?

using 0.2um nitrocellulose thinking of changing to PVDF, will this help?

-Timothy Bredy-

Hi Timothy:

I am new to this forum. I am writing to you because I am working also with histones (from Yeast S. Cerevisiae). I am purifying histones and then trying to look at acetylation by Mass Spec. I am having trouble to found a reliable anti-acetyl Ab. We tried one Ab from Upstates ( cat # 06933) but in only recognize H4 (acetylated I guess) and then we change to one Ab from Abcam ( cat #Ab 21623) and it seems to be very specific for acetylated BSA.

I am using butyric acid for the homogenization buffer and then performing a sonication step of the nuclear fraction in 6M guanidine hydrochloride , so I believe that deacetylation should not be a problem in my case.

As this an old posting I do not if you still have a problem detecting H4 (I guess not), so if you already find a solution , can you recommend a good commercial Ab for acetylated histones and give some details on your experience working with it.

Thanks, sorry if this issue is not longer relevant for you

Ernesto


QUOTE (Timothy Bredy @ Aug 31 2005, 10:11 AM)
Hey there,

I am doing western blot for acetylated histones 3 and 4. H3 works well, but I cannot get a signal for H4. Have tried 2 different Abs and several concentrations.. nothing. I even loaded 40ug protein, still nada!

Any suggestions?

using 0.2um nitrocellulose thinking of changing to PVDF, will this help?

-Ernesto-