Ac-H4 western - help! (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, are you seeing a band(s) of unexpected size? if you were then as annat says, you need to acid extract. If you are seeing no bands at all, maybe your antibodies are not there an not binding anything.

Good luck

Nick

Definitely, its easier to perform westerns on acid extracted histones. There is an easy protocol at:

http://www.abcam.com/index.html?pageconfig...rotocol&pid=316

Youll be able to see all the histones by Ponceau when you transfer them.

I have used Abcams Ac-H4 abs to good effect on WB, theyre great.