Western blot is not working with stable cell line=please help - no expression of my protein detected (Jun/03/2008 )

For last couple of months i have been creating a stable transfectio of the transcription al fcator, I selected my cells with G418 over 4 week s and still doing so , i started with 400mg/ml all way up to 800mg/ml. Upon inspection i saswe clones and then split cells and let them gro w a bit more, now i need to analyze the expression of targeted gene via western. My gene has HA tag so i use HA an tiboty for western So here is the problem, muy transient transfection i can see the expresion of the all my clones but wneh i check the stable transfected ones i dont see any expression, and i am wodering if something is worng with western or i just dont have expression on those stable cell lines. please help cause i dont know what to do. If there is no stable trasnfection whta make the cells survive the under the G418.
1st timethe film was blank even though i saw bamd with poncou abd i detect the b-actin

Sometime stable transfectants stop expressing your gene on interest, while still expressing the resistance marker. In my experience, transient transfections always gave higher expression than stables. High expressing cells will be selected against, if your gene is going to affect cell growth.

If you really need a stable transfectants, you should isolate as many clones as possible (>20) and maybe one or two will give you detectible levels of protein. You could also try to clone into an inducible vector, if your gene may be toxic.
-dan

Pretty good chances. Even stable cell lines that do express can all of a sudden stop expressing. It can be frustrating to make a cell line, freeze it, then re-use it later and find it doesn't work anymore. Maybe someone else on this board has protocols that are more reliable. In my experience, getting 1/20 clones was good, but I'd try to use it for experiments as soon as possible (and always making sure to freeze aliquots away, in case the expression stopped a few passages later). I am all for transient transfections for most experiments.