I want to do some pulse-chase protein degradation experiments in yeast. I
have done this in mammalian cells but I have a few problems with yeast.
If I label with 35S-methionine , wash the cells in medim containing
unlabelled methionine and then add TCA to 10%, I find that even without any
"chase" there is still a lot of TCA soluble radioactivity left (10% of TCA
insoluble). Any ideas why this may be so? Does centrifugation (2000g)
stress the yeast so they degrade protein faster, or is it difficult to wash
out the precursor? Is it possible to filter the yeast in some way, wash
them on the filter and then resuspend them in fresh medium and grow them
on? If so what kind of filter do I need?
Fergus.Doherty at nottingham.ac.uk
0115 970 9366 (74-41366 internal)