I wanted to confirm the binding of a lncRNA to a known protein. The protein is mostly disordered except for the part that may be involved in the binding (a coiled-coil region at the end). I was thinking of performing an EMSA to confirm the binding. And then maybe I was thinking of doing a CLIP-seq to delve deep into the binding motifs and sites to see if the RNA binds to the binding domain or some other site of the protein.
My question is - is CLIP-seq something that one can perform after establishing a binding between two components? As far as I know, CLIP-seq can give more detailed and confirmed binding data concerning motif and site-based proof of the RNA binding to the protein. If not, what can be some other in-vivo experiment that can be done to check the nucleotide-level binding of the RNA-protein?
Hi Haimantika.
EMSA is being used to detect protein-nucleic acid complexes. However, with your protein being disordered, it may not migrate as a single band, and also your lncRNA may be too long for this type of analysis, complicating interpretation of the results.
If you want a relatively quick experiment, you my use your protein of interest as a bait (e. g. FLAG/His-tagged, bound to beads) and test whether your lncRNA binds to it (include also a different RNA as a negative control). You can then detect/quantitate your lncRNA by various approaches (e. g. RT-qPCR).
Best,
Libor
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