3 replies to this topic

[Dave_Kub_11]

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.co...t.2007.202.html

Thanks,

Dave

[sBrow]

Dave_Kub_11, on 10 May 2011 - 02:08 AM, 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.co...t.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

[Dave_Kub_11]

sBrow, on 10 May 2011 - 03:29 AM, said:

Dave_Kub_11, on 10 May 2011 - 02:08 AM, 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.co...t.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.

[TheGreven]

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.