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?
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
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?