Transfection of expression vector for gel shift - how much vector should be transfected? (Mar/20/2007 )
I'd like to do my first gel shift assay and therefor want to overexpress the nuclear protein of interest in HEK293 cells first (to use this cell extract for the EMSA later).
I'm wondering how much expression vector I should transfect (cells in 100mm plate) to get an appropriate amout of protein for the gel shift. In one paper I read they transfected 15µg of vector/100mm plate. That sounds like a lot to me.
Has anyone experience with similar gel shifts and can give me some hints? I would greatly appreciate this :-)!
Its about right. You can use 15-25 ug, depending on the reagent you use. Usually such info is included in the product insert. I use up to 4 ug DNA per well for 6 well plate. That translates to ~20 ug / 100 mm dish.