Materials and Reagents

Oligo DNA 5’ end-labeled with IRDye 700 or IRDye 800 (sense strand) (Integrated DNA Technologies)
Non-labelled oligo DNA (both sense and antisense strands)
Non-labelled mutated oligo DNA (both sense and antisense strands)
Recombinant DNA-binding proteins expressed in Escherichia coli (E. coli) (10 ng/µl in protein storage buffer)
Sterile distilled water (SDW)
Odyssey infrared EMSA kit (LI-COR, catalog number: 829-07910 )
Poly(dI-dC) (Sigma-Aldrich, catalog number: 4929 )
Tris
Boric acid
EDTA 2Na
NaCl
HCl
Acrylamide
N,N’-Methylene-bisacrylamide
Glycerol
Triton X-100
Phenylmethylsulfonyl fluoride (PMSF)
β-mercaptoethanol
Ammonium persulfate (APS)
N,N,N',N'-tetramethylethylenediamine (TEMED)
10x TBE( see Recipes)
4% native polyacrylamide gel (see Recipes)
Native-PAGE running buffer (see Recipes)
Protein storage buffer (see Recipes)

Equipment

Procedure

Suspend lyophilized oligo DNA with dH₂O and mix them as follows (final concentration is 50 µM each).
Probe: labelled and complementary non-labelled oligo DNAs
Competitor: non-labelled and complementary non-labelled oligo DNAs
Mutated competitor: mutated non-labelled and complementary non-labelled oligo DNAs
Heat at 100 °C for 10 min on heat block for denature.
Turn off heat block and leave denatured DNAs on the block until room temperature.
Dilute probes to 50 nM with dH₂O.
Prepare 4% native polyacrylamide gel and 0.5x TBE.
Pre-electrophoresis for 30 min at 150 V at 4 °C.

Prepare reaction mixtures as follows during pre-electrophoresis:

Note: Tube numbers indicate the vial numbers in Odyssey Infrared EMSA kit.

Place at room temperature for 20 min in dark.
Note: Avoid light during reaction not to reduce signal intensity.
Add 2 µl of 10x Orange Dye (tube 10) to reaction mixture after reaction.
Wash gel wells with electrophoresis buffer (0.5x TBE) after pre-electrophoresis.
Load the samples on gel and run the gel at 150 V for 2.5 h at 4 °C in dark until the Orange Dye migrates to the bottom of the gel.
Remove gel from glass plate and place it directly on Odyssey.
Adjust focus offset of Odyssey to 1/2 of gel thickness and scan.
Note: Carefully remove air bubbles between gel and Odyssey.
Look at supplemental Figure 19 of Reference 1 as a representative EMSA result.

Recipes

Acknowledgments

This protocol is adapted from Nakata et al. (2013).

References