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An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides
Published on: November 29, 2016
Elimination of nonspecific bands in non-radioactive electrophoretic mobility shift assays using the digoxigenin
Saimi Tokunaga1, John J Stegeman1
1Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA.
Abstract:
In the course of detecting nuclear transcription factors by electrophoretic mobility shift assay using digoxigenin (DIG)-labeled probes, we encountered a problem with a considerable nonspecific shift band in negative control lanes from which protein extracts were omitted. This nonspecific shift band can interfere with the detection of the desired target protein. Purification of the DIG-labeled probes by removing unincorporated DIG-labeled nucleotides did not resolve the problem. However, the introduction of an additional step of heating at 95 °C for 5 min and subsequent reannealing after DIG-labeled probe synthesis eliminated these nonspecific shift bands and allowed accurate analysis of the target protein.
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