[Protein-analysis] Re: proteins in urea-SDS

Vinod Agarkar via proteins%40net.bio.net (by agarkarvi from yahoo.com)
Sat Mar 10 06:44:53 EST 2007

>Try guanidium chloride instead of urea
>Try alkaline pH, even to run a gel
>50mM DTT is ok
>Heating samples at 70 C for 5 min
All the best, Vinod

  In article , kaj.stenberg from helsinki.fi.invalid wrote:
>Florencia Campetella wrote:
>> Hi, I solubilized a pellet with a buffer containing 70 mM Tris-NaOH [pH 6.8],
>> 8 M urea, 2.5% SDS, 0.1 M DTT, 10% glycerol, bromophenol blue, and kept the
>> samples on ice. When going to load the gel I noticed some aggregates I
>> couldn't get rid off, gelatin-like. It's the first time I'm using this
>> buffer so I don't know what could be happening...
>> I was told urea is not stable and over time it ionizes and really affects
>> your protein sample, can anyone tried to freeze their samples with this kind
>> of buffer?
>8M urea???
>What is the rationale for that? 

Some proteins don't unfold completely in SDS, needing additional 
denaturant. Urea is a non-ionic denaturant so for some applications
it can be an advantage. 

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