I am using other plasmids (e.g. C1/2/3/4) that may aid in interaction of the two proteins. However, the level of expression of plasmids (C1/2/3/4) is significant if I transfect same amount of plasmid. Hence I am unable to determine their effect on protein interaction. what should I do next? is that feasible if I make their expressions (C1/2/3/4) in the similar level? And how to make their expressions in the same level. Is there any paper that uses this method? Thank you very much
What about the use of bicistronic vector where two genes are expressed from the same vector?
If you are in eukaryotic cells - as suggested by the term "transfect" in your query - you have the option (as suggested by Dr. Ali) of a bi-cistronic vector, or alternatively using an IRES or a FMDV2A sequence depending on exactly what you are trying to do. In each case, more than one product can be driven off the same induction stimulus so the proteins may be induced stoicheometrically.
Hi there! I'm a bit confused about the connection you make between expression levels and protein-protein interactions. No matter how much you can fine-tune the relative expression levels of each construct, this still doesn't tell you anything about interactions, correct? Maybe your question could use some clarification, if I misunderstood.
Do the separate proteins behave well in isolation? If so, then I would say that you should purify them individually and test for complex formation by biochemical methods such as analysis of gel filtration peak shifts or affinity pull-down assays. I'm skeptical of claimed interactions unless I can see it occurring with purified protein. It can be quite a burden of course, but there is currently somewhat of a crisis in bioscience when talking about putative protein-protein interactions... too much sketchy & ambiguous data in the literature!
Cheers, and good luck!
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