Hi fellow yeast addicts,
I have a S. pombe strain with a pREP1 plasmid that express a
secreted soluble protein. After induction in EMM ade+ ura+, HEPES 50mM,
pH 7, ammonium sulfate is added to the medium (minus the yeast) to a 80%
concentration to precipitate the secreted proteins. After the an O/N
dialysis at 4 Celsius in HEPES 50mM, NaCl 50mM, pH 7, I have a clear
supernatant but I always have a white insoluble precipitate. I suspect the
precipitate to be a protein/calcium or phosphate salt complexe. Is this
problem common when using EMM? If so, what sould I do to prevent it?
Have a good day,
D.H. Vuong, M. Sc. student
Biochemistry Dept. University of Montreal
P.S. Final concentration of CaCl2 is 0,1mM and Na2PO4 is 15,5mM.