we are studying E2Fs expression on a different stages of cell cycle progression. E2Fs change their intracellular localization depending on phase of cell cycle they are in. we have very good results in overexpressed experiments, but on endogenous level protein expression is not high enough to detect the difference by immunofluorescence (western blotting). We are trying to enrich population by cell synchronization and then releasing and collecting on the different stages. But it's still not enough, it would be great if one would be able on top of synchronization perform a cell sorting. But the problem is that for PI staining I need to fix cells and I am not sure that one can get cyto and nuclear protein fractions after fixation. E2fs are pretty small molecules and most probably will escape from the nuclei. I am looking for some staining procedure which will allow me to learn a phase of cell cycle what cell are in any particular moment so I can sort them out and after that separate cyto and nuclear protein fractions. Thank you very much. Marina -- Marina Polonskaia, Ph.D. SUNY@Stony Brook tel.: (631)632-8818 e-mail: mpolonskaia@yahoo.com marina.polonskaia@sunysb.edu
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