Hi Marina, Interestingly, we have been trying to do something similar to look at subcellular localisation of protein (in a fairly broad nuclear v cytoplasmic way) and correlate that with cell cycle phase. The approach we have taken is to stain the cells initially with Hoechst 33342 (10ug/ml, 45mins at 37C but this may vary with cell type so you might have to optimise the conditions) - this will quantitatively stain the DNA without the need for fixation - I can then sort according to DNA content into G1, S and G2/M (I use UV excitation - thats 351-363nm - on a Cytomation MoFlo and can sort all three fractions simultaneously). You could then take the cells for protein extraction and Westerns. If you dont have access to a UV source a potential alternative is DRAQ5 (http://www.biostatus.co.uk/draq5.html). What we have done though is put the cells onto a slide, fix and stain for the protein of interest with a FITC-labelled antibody. The cells are then counterstained with propidium iodide and analysed on Laser Scanning Cytometer (http://www.icnet.uk/axp/facs/davies/lsc.html). By thresholding on nuclear fluorescence but also integrating an area around the nucleus, we can get an idea of the expression of the protein within the nucleus and cytoplasm. Its early days yet but what we are hoping to see is changes in the levels of the protein in the compartments after particular treatments. Hope that helps and good luck! Derek On Tue, 18 Dec 2001, Marina Polonskaia wrote: > 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 ************************************************************************ Derek Davies Voice: (44) 020 7269 3394 FACS Laboratory, FAX: (44) 020 7269 3100 Imperial Cancer Research Fund, e_mail: derek.davies@icrf.icnet.uk London, UK mobile: 07790 604112 Web Page: http://www.icnet.uk/axp/facs/davies/index.html In tenebris lux *************************************************************************
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