I have this Glioblastoma cells line transduced with viral soup made from lipofectamine 3000 (containing desired plasmids,ppax and pmd2G.) what is this black things growing in background and how to get rid of this??
Has anyone performed FRAP on a confocal microscope using Alexa Fluor secondary antibodies to study colocalisation? I already did standard immunofluorescence colocalisation analysis, but I’m wondering whether FRAP would provide any additional useful information in this setup. Most papers I found use GFP-tagged proteins instead of secondary antibody staining that is basically FRET i guess. Would appreciate any advice or relevant references.
I wanted to perform immunoprecipitation from the nuclear fraction of paraformaldehyde-fixed cells to preserve protein–protein interactions.
Manual subcellular fractionation works well in normal/unfixed cells, and under the microscope the cells appear lysed properly. However, with PFA-fixed cells, lysis has become extremely difficult — even harsh lysis buffers are not working efficiently.
Has anyone worked with nuclear extraction/IP from fixed cells?
Any suggestions would be really helpful.