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Updated: Jan 12, 2026
An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides
Published on: November 29, 2016
An electrophoretic mobility shift assay with chemiluminescent readout to evaluate DNA-targeting oligonucleotide-based
Michaela E Everly1, Peter J Wieber1, Ibrahim Al Janabi1
1Department of Chemistry, University of Idaho, Moscow, Idaho, United States of America.
None:
A comprehensive and user-friendly method for evaluating recognition of double-stranded DNA (dsDNA) targets by oligonucleotide-based probes is presented. Thus, dsDNA-targeting probes such as single-stranded locked nucleic acids (LNAs) and double-stranded Invader probes are incubated with digoxigenin-labeled DNA hairpin targets, and the resulting recognition complexes are resolved using an electrophoretic mobility shift assay and tagged using a chemiluminescence immunoassay. Emissive products are detected by a C-DiGit Blot Scanner and quantified with the accompanying software. R-based scripts for data visualization and determination of C50 values (a measure of the dsDNA-binding affinity of a probe) are also provided. The data presented here demonstrate the effectiveness of the described protocol and highlight the variable dsDNA-recognition efficiencies of LNAs, Invader probes, and chimeric Invader:LNA probes.
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