I am trying to replicate the FP assay from the following publication and am wondering why the change in mp value between RNA(70-nt long, 23 KDa) and the fluorescent-peptide (12-mer, 2 kDa) is very low (as can be seen on page 8 of SI document) despite the large size difference between the two molecules. Based on my reading and experience of FP between a protein and a probe peptide of similar size, the mp change is on the order of 80-100 mp. Any thoughts/suggestions are appreciated.
http://www.nature.com/nchembio/journal/v7/n8/extref/nchembio.596-S1.pdf
Article Discovery of Selective Bioactive Small Molecules by Targetin...
Thanks Theo for your suggestion. It is an interesting idea. I can image having the biotin and streptavidin complex on RNA would make it much bulkier. My problem with this assay is the noise and signal instability (varying kd values) over time which I am hoping to improve by increasing the dynamic range of the assay.
I agree with Theo that the range of polarization values should be sufficient given the inherent precision of polarization measurements. The main reason that polarization values do not increase as much as expected from the molecular weight change is due to local probe mobility. In other words, if the fluorescein moiety if free to rotate around its linkage, when the peptide is bound to the RNA, then the polarization can be substantially lower than expected.
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