Transfection - (May/02/2001 )
i am trying to express a cloned gene in mammalian cells.i have tried 3 different cell lines and two different methods also.one is calcium chloride and the other is QIAGEN trasfection reagent EFFECTENE.after trasfection i am doing a western to confirm the presence of protien but i am not getting any signal..toghether with this i am using a GFP vector as a reporter.there also i can see only very few cells expressing GFP.can anybody out there give me any suggestions?
First: Why is your promoter and Are in your cell type the necessary factor for your expression? For example: SV40 will need T Antigen.Second: Are you doing stable or transient transfections?
Effectene should be quite good for a wide range of cells. The problem may be your plasmid constrution, or inappropiate GFP control. Try clontech's pEGFP-N1.