Nuclear lysis followed by histone extraction - (May/10/2011 )
I intend to extract nuclear proteins from my cells and then purify histones from the chromatin pellet. At the moment, I plan to acid extract with 400ul 0.2M H2SO4overnight then precipitate out the histones from the supernatant with ice-cold acetone.
Does anyone suggest how much acetone is necessary to produce a good yield of histones?
Also throughout the lysis, should I supplement EVERY buffer with sodium butyrate to inhibit deacetylation? At the moment I have it in my first 2 lysis buffers.
I'm generally follwing this protcol for anyone interested: http://www.nature.com/nprot/journal/v2/n6/full/nprot.2007.202.html
Thanks,
Dave
Dave_Kub_11 on Tue May 10 10:08:49 2011 said:
I intend to extract nuclear proteins from my cells and then purify histones from the chromatin pellet. At the moment, I plan to acid extract with 400ul 0.2M H2SO4overnight then precipitate out the histones from the supernatant with ice-cold acetone.
Does anyone suggest how much acetone is necessary to produce a good yield of histones?
Also throughout the lysis, should I supplement EVERY buffer with sodium butyrate to inhibit deacetylation? At the moment I have it in my first 2 lysis buffers.
I'm generally follwing this protcol for anyone interested: http://www.nature.com/nprot/journal/v2/n6/full/nprot.2007.202.html
Thanks,
Dave
Hi
I have done loads of histone extraction. successfully...using only a single lysis buffer. I can send you the recipe and protocol? we use 1mL actone and leave overnight in fridge...then wash 2x with another mL..then resuspend in 25-50uL buffer...
my home email: nzsusanna@yahoo.co.nz
cheers
sBrow on Tue May 10 11:29:58 2011 said:
Dave_Kub_11 on Tue May 10 10:08:49 2011 said:
I intend to extract nuclear proteins from my cells and then purify histones from the chromatin pellet. At the moment, I plan to acid extract with 400ul 0.2M H2SO4overnight then precipitate out the histones from the supernatant with ice-cold acetone.
Does anyone suggest how much acetone is necessary to produce a good yield of histones?
Also throughout the lysis, should I supplement EVERY buffer with sodium butyrate to inhibit deacetylation? At the moment I have it in my first 2 lysis buffers.
I'm generally follwing this protcol for anyone interested: http://www.nature.com/nprot/journal/v2/n6/full/nprot.2007.202.html
Thanks,
Dave
Hi
I have done loads of histone extraction. successfully...using only a single lysis buffer. I can send you the recipe and protocol? we use 1mL actone and leave overnight in fridge...then wash 2x with another mL..then resuspend in 25-50uL buffer...
my home email: nzsusanna@yahoo.co.nz
cheers
What I should've have stressed is that I use 2 lysis buffers and a wash buffer in the nuclear lysis procedure and I was wondering if I should include sodium butyrate in all of them.
Hi,
I have a follow up question about acid extraction of histones. What is the advantage of acid extraction of histones if you just want to do a Western blot to check modifications compared to just lysing your cells with a SDS based lysis buffer (or SDS sample buffer)?? The only advantage I see is that you get a more pure sample. Other than that I dont see any other advantages. Also, acid extraction takes longer time since you have an overnight step. With a regular SDS lysis buffer you just add it to you cells, incubate 15-30 min on ice (optional: sonicate briefly), spin down and collect supernatant which contains your proteins.
I would be very happy if someone could give me an answer.