I am trying to replicate a colocalization experiment but I am having no luck. The only difference between the previous experiment and my own is that we are using different transfection reagents: superfect vs. lipofectamine. Could the use of a different transfection reagent prevent the colocalization of two proteins? These are both membrane proteins.
As I know transfection reagents won't affect the protein interaction. You use the trasfection reagents to deliver your target DNA (should in a plasmid?) into cells and then, after a sequential process, your DNAs are translated into proteins. The transfection reagents are no longer there with your proteins. Usually transfection reagents have affinity to DNA or RNA, not protein. They form a complex with DNA, which is only stable for like 6 hrs.
But since it's not a big deal, why not just use superfect for your experiment?
Maybe the interaction is not that real? It's something mean to say but often it happens!
Different tranfection reagents need to be optimized differently. And to my experience, they do not influence in vivo protein interactions. But to be absolute sure, you should have a control with only transfection reagent to see, if there is any reaction.
They should not affect protein / protein interactions directly, though some reagents can add more stresses to the cells which may cause changes. You can try washing out the transfection reagent sooner to decrease the stress on the cells. Another thing I like to do for my transfection experiments is pass the cells onto coverslips the day after transfection. This gives them more time to recover from effects of transfection and also slow down protein production to more modest levels. In general it allows for more uniformity between experimental repeats and might help your situation here.
Another thing is are these live or fixed cells you are assaying? Because different batches / qualities of fixative can lead to changes in fixation efficiency and distort/expand intracellular membranes, which may lead to different results, especially in a sensitive colocalization assay.
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