Mobility shift assay protocols can be easily converted to infrared fluorescent assays by replacing the existing DNA oligonucleotides with IRDye® infrared dye end-labeled oligonucleotides. Binding and electrophoresis conditions are the same as any other EMSA detection method.
The Odyssey EMSA Kit, coupled with IRDye 700 EMSA oligonucleotides, is an excellent alternative method to radioisotopic and chemiluminescent detection methods for EMSA analysis and visualization1, 2. Using IRDye EMSA reagents, assays can be completed in less than two hours with no gel transfer or film exposure. The gel doesn’t even need to be removed from the glass plates for imaging.
- 10X Binding Buffer (100 mM Tris, 500 mM KCl, 10 mM DTT, pH 7.5), 500 μL
- 25 mM DTT, 2.5% Tween® 20, 500 µL
- Poly (dI•dC), 1 µg/µL in 10 mM Tris, 1 mM EDTA, pH 7.5, 125 µL
- Sheared salmon sperm DNA, 0.5 µg/µL in 10 mM Tris, 1 mM EDTA, pH 7.5, 125 µL
- 50% Glycerol, 500 µL
- 1% NP-40, 500 µL
- 1 M KCl, 500 µL
- 100 mM MgCl2, 500 µL
- 200 mM EDTA, pH 8.0, 500 µL
- 10X Orange Loading Dye, 500 µL
Example of an NIR Fluorescent EMSA
- Li, Y., et al. (2005) Cancer Res. 65: 6934-6942. DOI: 10.1158/0008-5472.
- Geddie, M.L., et al. (2005) J. Biol. Chem. 280(42):35641-6. DOI: 10.1074/jbc.M508149200