how to improve co-transfction efficiency? - (Jan/28/2007 )
in my expriment ,two plasmids were co-transfected into cos7 cell,followed by CO-IP assay,the problem is co-transfection efficiency is too lower,i can't get good CO-IP result,how to improve the co-transfection efficiency?
any suggestion is appreciated.
thanks
Well, you need to optimise your transfection method. How do you transfect your cells?
also its important for Co-IP to have good antibodies. So also check the antibodies.
also its important for Co-IP to have good antibodies. So also check the antibodies.
hi, i using lipofectamin2000 to transfect cos7 cell, tansfection were done as this product protocol introduction.briefly,first day cos7 cells were plated and culture overnight , transfection were done in 20h after the cells were plated, one plasmid is myc-tagged and the other is HA tagged, and both the plasmid concentration is good(>1000ng/ul), I using anti c-myc to precipetate and anti-HA to detect the interested protein, always i can't get result. I wonder the co-transfection efficiency is the key problem, if the most cells just only have a single plasmid, the interaction would not happen.
could you give me some suggestion to optimise my transfection method,thanks.
To make sure co-transfection is efficient, use 2 different fluorescent protein reporters and see how many cells take up both plasmids. Have you checked single transfection to see if your antibody's are working and how efficient at least single transfection is? Optimisation would be to play around with ratio's of DNA vs volume of transfection reagent. Apart from this, using Opti-Mem to dilute DNA and LF2k gives better results than other media, maybe that's worth a shot?