Luciferase assay - experimental design - (May/17/2016 )

Hi all,

I'm designing an experiment to determine if a combination of five transcription factors is required for transcription of my target gene. I'd like to perform a western on my transfected cells in addition to the luciferase assay so that I can make sure that the transfection worked for all five factors. Normally, the lab does the assay in 96-well plates and doesn't confirm that the transfection worked, which I do not like. I'm looking for suggestions on how to go about this. Ideally, I'd like to transfect my cells (adherent) in a 6 well plate and use the same cells for luciferase and western. However, given that the lysis for the luciferase activity is done at room temperature, I don't have high hopes for a successful western. Any tips/strategies are greatly appreciated!

Thanks,

Sarah

There are a number of luciferase reagents that can be used in regular lysis buffers, You should be able to do the lysis for the luciferase in the cold, but it will take longer. You could also lift the cells, wash, resuspend and then take an aliquot for luciferase and another for WB.