8M urea buffer for protein extraction - (Oct/09/2008 )
I would like to prepare protein extranction buffer containing 8M urea.
Do I need to autoclave this solution before use?
No. 
No, you don't need to autoclave. But be aware that some protein quatification methods find very hard to measure protein concentrations in such a high urea concentrations...I had a coulple of troubles using 6M urea with 2 M thiourea for extraction buffer..my BCA anaylis went crazy.....all I could measure was urea....
good luck.
do not autoclave urea, it will break down.
if you need to sterilize a urea solution then do it by filter sterilization.
I have another question - I separate membrane / soluble proteins using Prot-Two Kit from Sigma. My final samples are dissolved in 40 mM Tris, 7M urea, 2M thiourea and 1% C7BzO detergent, pH 10.4 (and they have already been treated with TBP/IAA). Do you think that these extracts are compatible with SDS-PAGE/Western blotting?
First, I was wondering about pH - I'm afraid if I mix those samples (pH 10.4) with my 6X sample buffer, the final pH value will hardly be 6.8, won't be? Is correct pH of sample critical for running and protein resolving?
Second, is it possible to heat those urea-based buffers as I do in 'standard' protocol to enhance protein denaturation (65C, 15 min)? In the product info, there is written that these buffers should not be heated above 30C to avoid formation of cyanates which could damage proteins.
Is the combination of urea/thiourea/detergent at above mentioned concentrations enough for effective protein denaturation (so there is no need to heat samples), or could be better denaturation achieved with heating in the presence of e.g. SDS?
If I decide to precipitate proteins (or maybe ultrafiltration could do a good job as well?), what would you suggest for protein solubilization? Just 1x sample buffer for SDS-PAGE? (I don't need to measure protein concentrations, so bromophenol blue would not interfere)
yes.
pH is important. you should ensure that your sample ends up near 6.8.
Is the combination of urea/thiourea/detergent at above mentioned concentrations enough for effective protein denaturation (so there is no need to heat samples), or could be better denaturation achieved with heating in the presence of e.g. SDS?
you should not heat the protein in the presence of urea. you can add the loading buffer and let it sit at rt for a few minutes before loading.
yes.
thank you, I tried it and it worked nicely in the original urea-buffer (+1/6th volume of 6X SDS-PAGE sample buffer) w/o heating and I got the same results when I had ultrafiltered my samples, recovered to 1X SDS-PAGE sample buffer and heated.