Dear all, Thanks for all the replies. I adjusted my volumes and stopped reactions with 8 M urea and 25 mM EDTA. The gels now run fine :)
-Mohammad On 05-Oct-2017 7:29 PM, "Phoebe A. Rice" <pr...@uchicago.edu> wrote: > Even though the protein should be denatured by all that urea, we find such > gels sometimes look nicer if stop the reaction with SDS and protease K, > and/or phenol extract the remains of the protein before loading the > high-urea gel. > > ++++++++++++++++++++++++++++++++++++++++++ > > Phoebe A. Rice > Dept. of Biochemistry & Molecular Biology > The University of Chicago > pr...@uchicago.edu > > > ________________________________________ > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Opher > Gileadi [opher.gile...@sgc.ox.ac.uk] > Sent: Saturday, September 30, 2017 3:44 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Off topic: denaturing urea gels > > In addition to the previous suggestions: > > With very small gels, the sample composition and depth (in the well) have > a strong effect on the resolution. > Rinse the wells with TBE buffer just before loading, as urea from the gel > diffuses into the well and may prevent the sample from settling at the > bottom. Minimize the amount of salt in the samples; try to load very small > volumes (1-3 ul), even if this means longer exposures later.
