cell culture with serum media and with serum free media and doubts in luciferase - (Jun/12/2007 )

Dear all, I am having some doubts with luciferase assay. I am doing luciferase assay with human 3T3L1, 3T3L6 and HepG2 cells. I am also doing protein assay and calculating luciferase results with protein. Till now i have repeated 3 times with each type of cell and I am getting different results each time (different expression levels). I am thinking that my calculation with luciferase and protein is not correct. can any one please tell me how to do calculation with luciferase expression and protein concentration to get the exact luciferase activity. For transfection i am using promega fugene6, serum free media and DNA of concentration 1microgram /microleter. I am having doubt that whether i am using right concentration or not? Can you please suggest me what is the right concentration of these for single well in 12 well plate.

Now I am also thinking to add the insulin to serum free media and check the expression. After how many days or hours I should add insulin to serum free media after transfection?

At last i also want to cotransfect beta gal with luciferase. How to do this cotransfection and what is the correct procedure? It will be better for me if any one can send me protocol for preparing cell lysate from cotransfected cells. It will be easy for me if any one can provide complete protocol beginning from culture of above cells, transfection and preparation of cell lysate for luciferase assay and protein assay and beta gal assay.

I will be very thankful for all of u for having patience for solving all my above doubts.

I think you are using too much DNA. I usually use 1ug in a 6 well so that would make 0.5ug for you. Too much DNA and the transfection isn't efficient. Also make sure that you dilute the Fugene in serum free media that also has no additives (e.g. antibiotics and glutamate). I usually do the transfection early afternoon and the following morning replace media, after 2h I add the treatment I want.