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good transfection efficacy, no proteins in WB - the weirdest problem of my life (Jun/04/2008 )

Hello everybody,

I'm a french Ph-D student and I have a big problem with protein expression. When I perform calcium phosphate transfection in HEK293T cells with EGFP (5µg for 10cm dishes), the expression is OK (seeing in WB and immunofluorescent microscopy) but when I transiently transfect my plasmids of interest (coding for 2 differents flagged proteins) I see nothing after Western Blot analysis. And I perform this type of transfection and WB since 2 years with success (the same plasmid type but from a different maxiprep). The weirdest thing is that I have performed lots of transfection and WB since the 2 past months and my WB analysis worked perfectly 2 times (detection of my 2 flagged proteins). So the week after, I kept exactly the same condition, I also transfect an EFGP dish to ensure transfection efficacy. It was OK. But when I performed WB I detected only EGFP!!? I'm performing tests since 4 months and I'm become crazy. The plasmids looks ok on an agarose gel electrophoresis, the sequence is OK, my technique of nuclear extraction seems good (detection of EGFP in WB) and my WB techniques and antibodies (@FLAG) seems OK because I can easily detect my transfected proteins in older nuclear extract. So please help me before I change my job from biological research to pizza seller.



The only way to get through a problem like this is to do controls.
#1. Throw out your 293T cells and get a fresh batch.
#2. To determine if technique is flawed, transfect an expression plasmid for a different protein. Can you detect this by Western? If so, your technique is fine but maybe your FLAG plasmid is bad.
#3. Is you anti-flag antibody gone "off"? Transfect another FLAG tagged protein to test this antibody.
#4. Do a new MAXI-PREP.
#5. Get new transfection reagent.

Something you are doing isn't working. Either the plasmid, the transfection or the
antibody isn't working so you need to systematically rule out each possibility.