[RASMB] Protein degradation in AUC

[John Burgner]

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



-- 
Prof. Dr. Claus Urbanke
Medizinische Hochschule
Abtlg. Strukturanalyse OE 8830
Carl-Neuberg Str. 1
D-30625 Hannover
Tel.: +49 511 532 9372
FAX:  +49 511 532 5966

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