I just wanted to check the nuclear translocation of a transcription factor in response to my drug treatment in cell lines ?
What would be the best technique to study this ?
I think the easiest way would be a translational fusion with GFP or any other fluorescent protein, otherwise, if you have antibodies against your transcription factor, immunolocalization would be a good option.
For the use of fluorescent proteins: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2875081/
You must have a good antibody. If so, the immunofluoresence in cell culture is the best method. This can then be confirmed by SDS/PAGE and western blotting of nuclear and cytoplasmic factions. If you do not have a good antibody, then you must make either an epitope tagged form (flag, myc, or HA) in an expression vector or as suggested above a GFP fusion protein.
The expression vector is then transfected into your cells in culture. Make sure you do not overload the cells with too much plasmid. Try and express the protein at near physiological levels.
Western Blot on nuclear and cytoplasmic fractions is the most quantitative and physiological experiment.
Thanks fellas for the suggestions !!
i hope i can quantify the immunofluorescence in the nucleus , before and after the treatment
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