Protocol for histone acid extraction for Western blot - (Aug/22/2005 )
i think we need extract the histone specially. coz we do WB for tri-meth H3 K27 but got some strang band( looks like uneven loading but cann't see any different in actin).
so this time we extract nuclear seperately ,but i am not sure we need just extract hisone for methy-histone?
Hi acid extraction works well, just spin down after to remove non acidic proteins. To the supernatant I add 6 vol of cold acetone at -20 overnight then spin down the histone will precipitate, doing preps this way you get clean samples. I have done differant H3 meth a number of times now with good clean bands
I have recently tried acetone precipitation of HCL extracted 'histones'. I got a nice amount of precipitation.
I centrifuged at 12,000 rpm for 10 mins, discarded the supernatant. I breifly airdried the pellet, but not excessively (about a minute or two) and added lammeli buffer (with b-mecaptoethanol).
I have not been able to resuspend the pellet in lammeli buffer. I boiled at 100 degrees for a total of half an hour, even added unbelievlable loads of buffer in an attempt to get it to resuspend.
I have run an SDS PAGE gel of whatever was soluble and I get nothing!
Anyone got any ideas about how to resuspend the acid extracted histones following acetone precipitation?
Does my story sound familiar? Do people recommend buffer exchange/dialysis in place of precipitation for thins reason?
maybe you can neutralize the buffer with Tris or other base instead of TCA precipitation.