Western blot on FACS sorted Purkinje cells
We are trying to sort GFP Purkinje cells from all other cells in the cerebellum. We are having great trouble with this. Before going to the FACS facility we have 5 x 10^6 cells. The cells are passed through a 100 micrometer nozzle at a rate of 8000 events/ second. The sort decision has been based on both FSC, DAPI and GFP fluorescence. After the sort, which resulted in 800,000 cells, we have collected the cells in both media and PBS, spun the cells at 800rpm for 5 minutes and tried to run the samples on a Western blot and stain for GFP. We seem to be losing all GFP after the sort. Any help at all would be greatly appreciated.
Many thanks in advance,
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