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GFP-protein signal vanish - (Nov/18/2005 )

Hello there,
I hope this is the right forum and there is anyone out there who might help.
I'm overexpressing my GFP-tagged protein [Clontech C1-EGFP vector] (a kinase, function yet unknown) in HUVECS. I can detect and see the green cells after 6,8,10,and also still after 12 hours. Somehow after 24 hours there are only very very few green cells. Cells are alive and happy, but not green anymore. What might b the reason - any idea?
Thanks for thinking with me.


Just wondering, are you doing a transient transfection? smile.gif
For me, in transient transfection cells, the number of green cells decrease with time until 72 hours where there are very little green cells left.
I think for every vector, the optimal protein expression time period is different. Maybe after 24 hours is the time where the expression starts to decline. Maybe for you vector there is also a specification. If not, maybe you have to modify the conditions.
Is the time period sufficient for you assay?
If it is not, or if the cell population is too little for the assay,
maybe you would want to select a purer population of transfected cells overexpressing
your protein.
Hope this helps. smile.gif


Thanks, this is already something.
I'm doing transient transfection.i First I thought the disappearing comes from some sort of cell cycle dependency. But the promotor is CMV.