I am designing λN-BoxB tethering assay, my tethering plasmids are λN-RBPs and λN-RBP mutants (RNA binding motif mutations), I will carry on tethering assay to validate binding affinity between several RBPs and specific locations on RNA transcript. I am a little bit confused that since based on the tethering principles, recruitment of λN-protein was achieved by binding between λN and BoxB sites. Which means, the requirement of RNA binding motifs is not necessary, at least in those λN constructs, then, is it reasonable to use λN-RBP mutants as controls? I mean if binding was determined by λN and BoxB sites, should I expect difference between RBP-WT and MT? Please let me know your similar experience or correct me if I am wrong, or misunderstand anything about this technique.
Thank you so much!!
Lu Wei
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