I plan to introduce a human glutamate dehygrogenase in the brain of mice. Before that, I would like to validate my construction by infecting HEK cells. My construction CMV-GFP-2A- glutamate dehydrogenase mutant or wild type is in the shuttle2. How can I infect the HEK, how long? does anyone have a protocole to infect rapidly the cell? I thing that i will do after a western blot against both GFP and glutamate dehydrogenase and an immunofluorescence. Thanks a lot for your advices and help.
HEK293 are easily transfected with very high efficiency using either Lipofectamine or Fugene. Transfect your vectors into plates that are 50% confluent and harvest cells 48h later. Then make a protein lysate and do western blots. Even if transfection efficiency is on the low side, the CMV promoter is strong and you should see plenty of protein at the right size with specific antibodies.