Breaking yeast cells - (Sep/18/2007 )
I would like to open yeasts with glass beads in 8M urea. I know that urea is degraded when it is warmed up so I am wondering whether it is a good idea or not to use urea while I am braking cells with a bead-beater...
I assume you are going to extract denatured proteins? In which case, the extract should be rested every so often on ice to keep the mixture cold , while you are breaking the cells. Protein oxidation and protease activity (during the time that the protease inhibitors take to full inhibit all present proteases) would be the main concern... kept in check by low temperatures, DTT and previously mentioned protease inhibitor.
In any cause, urea doesn't break down easily. Below is a paper to examplify the point.
Degradation of urea in concentrated aqueous solution
Harry L. Welles, Alexander R. Giaquinto, Richard E. Lindstrom *
School of Pharmacy, University of Connecticut, Storrs, CT 06268
The degradation of urea in 2.00, 4.00, 6.00, and 8.00 M aqueous solutions was studied at 25.0, 35.0, and 45.0°. Data were obtained by measuring the specific conductivity of the solutions at 6-hr. intervals over 3.5 days. The results show that the degree of degradation is extremely small and that the overall process conforms to a first- and second-order reversible reaction. Rate constants were determined for the forward and reverse reactions and compare favorably to values reported by other workers for the separate reactions.
Thank your very much! Indeed I just want to extract proteins to digest them afterward so I don't care whether denatured or not... The only problem is that I cannot use SDS because of the downstream steps I have to do; thus I thought to use urea and I was just afraid that it could be degraded or do some side-reactions when it is heated up during the cell breaking... Thx!