I have Co-transfected HEK293 cells with equal amounts of two different DNA constructs. Both DNA sequences are present in pCDNA3.1+ vector background. When I performed western blots from the protein lysates, I observed that only one of the proteins is highly expressed whereas the other protein did not give any signal at all. I tested the antibodies before and they work good. What could be the reasons behind this low expression level. Is it due the difference in Kozak sequence in both DNA sequences?
On the other hand, I am planning to optimise the ratio of these DNA constructs to get good expression levels.
First question: did you try transfecting the plasmids separately (one plasmid in one transfection in one well and the next in another), so that you know may be the protein what you are not detecting may have nothing to do with your co-transfection(playing with plasmid ratios or transfection optimization would be secondary for me).
SEcond: how you are sure that the protein is not there? did you keep a positive control to see your antibody's activity?(in the same blot)..I would do this first to rule out any uncertainties,
May I know what is the need of cloning two different DNA constructs separatly in a separate pCDNA3.1+ vector ? you can Clone in as a single construct and express in HEK 293 cells? did you check whether low expressed gene construct alone can give expression ?
all the best
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