[RASMB] Protein degradation in AUC

[Dmitry Veprintsev]

Dear Claus,
in my experience some proteins are more prone to proteolysis then
others. Protein preps themselves can have proteases as impurities, and
cleaning the cells will not help.
In this case I found adding "Complete"  protease inhibitor cocktail from
Roche  very helpful.
regards, Dmitry

John Burgner wrote:
> Claus,
> We have used 6 M urea without any apparent problems.  I would suggest trying
> high concentrations of RNase Zap,  SDS, or DDM, which is what we do after
> using high protein concentrations with proteins that tend to strongly
> aggregate (pack at the bottom of the cell). The other approach that we have
> used is to put the centerpiece in a zip-lock bag with a detergent solution
> and sonicate it for 30 minutes then transfer the centerpiece to another bag
> with water and resonicate it for a few minutes.
> John
>
>
> -----Original Message-----
> From: rasmb-bounces at rasmb.bbri.org [mailto:rasmb-bounces at rasmb.bbri.org] On
> Behalf Of Claus Urbanke
> Sent: Friday, October 12, 2007 7:41 AM
> To: RASMB
> Subject: [RASMB] Protein degradation in AUC
>
> Hello RASMBers,
>
> for a few weeks now we encounter a nasty problem regarding the cleaning 
> of epon centerpieces (charcoal/aluminum). With an occurence of 10 - 30% 
> protein will be partially cleaved (by a protease?) after a sedimentation 
> run. Has anyone encountered this before? How can one clean the 
> centerpieces. The resistance chart tells me that GuaHCl is satisfactory 
> and urea is unknown, so we are testing some centerpiece fragments over 
> the weekend in concentrated urea solution.
>
> We are cleaning our centerpieces with a pipe cleaner using commercial 
> household detergent rinse them with hot water and finally with doubly 
> distilled (dd) water. The detergent itself was tested to be protease 
> free. We use saphire windows that are cleaned by >  1 hour 50% nitric 
> acid, rinsing with water, dd water and drying.
>
> Many thanks in advance for any suggestion
>
> Claus
>
>
>
>   

-- 
Dr. Dmitry Veprintsev
MRC Laboratory of Molecular Biology
Hills Road, Cambridge CB2 0QH, UK