2. Prepare a master mix solution (see Subheading 3.2). The final
concentration of nucleic acid probe should be low enough to
avoid ligand depletion (<10% K d ) (see Note 33) but high
enough to have good signal for detection. We typically use at
least 1–10 nM fluorescently labeled probe in a 10 μl reaction
(see Note 19).
3. Assemble the reaction, allow it to reach equilibrium (see Subheading 3.2; see Note 24), perform the EMSA, and image
the gel.
4. Open the image of the gel in image analysis software such as
ImageJ [16]. Change the contrast of the gel so that the bands
corresponding to free nucleic acid and protein-bound nucleic
acid are clearly visible (see Note 34).
5. Box each lane and measure the intensity of the band
corresponding to free nucleic acid by densitometry in the
image analysis software (Fig. 1c).
6. In a graphical analysis software such as GraphPad Prism
(GraphPad Software, Inc.), plot the intensity of the band
against protein concentration (Fig. 1d). The axis of protein
concentration can be changed to a logarithmic scale to aid
visual analysis. The concentration of protein at which half of
the nucleic acid is bound corresponds to the estimated dissociation constant (see Note 35).
3.6 Variation:
Supershift of Ternary
Protein –Protein–
Nucleic Acid
Complexes
Using EMSAs, ternary interactions can also be detected. If a prey
protein (P2) interacts with a bait protein (P1)-nucleic acid complex, it will cause a further reduction in electrophoretic mobility,
termed as “supershift.” The supershift assay can be used to demonstrate various aspects of the interaction. For example, the interaction of P2 with the P1–nucleic acid complex can be tested with P2
and nucleic acid alone. If P2 alone does not cause a shift in nucleic
acid mobility, then it must interact with P1 only, or at a composite
P1–nucleic acid binding site. P2 could alternatively disrupt the P1–
nucleic acid interaction (see Note 36). Moreover, if P2 is an antibody against P1, the identity of the protein P1 can be verified.
Finally, the supershift assay can also be used to assess the stoichiometry of a complex. For example, the ability of a protein to
multimerize on a nucleic acid substrate can be tested by varying
the length of the nucleic acid or the protein concentration.
1. Carry out a titration of P1 against the nucleic acid, as in
Subheading 3.5 above. Select a concentration of P1 where
the free nucleic acid band has completely disappeared.
2. Keeping the concentration of P1 constant, titrate an increasing
concentration of the putative binding protein P2 against the
protein–nucleic acid complex. It is important to keep one
sample with no protein as the negative control.
EMSAs to Detect Protein-DNA/RNA Interactions
329
concentration of nucleic acid probe should be low enough to
avoid ligand depletion (<10% K d ) (see Note 33) but high
enough to have good signal for detection. We typically use at
least 1–10 nM fluorescently labeled probe in a 10 μl reaction
(see Note 19).
3. Assemble the reaction, allow it to reach equilibrium (see Subheading 3.2; see Note 24), perform the EMSA, and image
the gel.
4. Open the image of the gel in image analysis software such as
ImageJ [16]. Change the contrast of the gel so that the bands
corresponding to free nucleic acid and protein-bound nucleic
acid are clearly visible (see Note 34).
5. Box each lane and measure the intensity of the band
corresponding to free nucleic acid by densitometry in the
image analysis software (Fig. 1c).
6. In a graphical analysis software such as GraphPad Prism
(GraphPad Software, Inc.), plot the intensity of the band
against protein concentration (Fig. 1d). The axis of protein
concentration can be changed to a logarithmic scale to aid
visual analysis. The concentration of protein at which half of
the nucleic acid is bound corresponds to the estimated dissociation constant (see Note 35).
3.6 Variation:
Supershift of Ternary
Protein –Protein–
Nucleic Acid
Complexes
Using EMSAs, ternary interactions can also be detected. If a prey
protein (P2) interacts with a bait protein (P1)-nucleic acid complex, it will cause a further reduction in electrophoretic mobility,
termed as “supershift.” The supershift assay can be used to demonstrate various aspects of the interaction. For example, the interaction of P2 with the P1–nucleic acid complex can be tested with P2
and nucleic acid alone. If P2 alone does not cause a shift in nucleic
acid mobility, then it must interact with P1 only, or at a composite
P1–nucleic acid binding site. P2 could alternatively disrupt the P1–
nucleic acid interaction (see Note 36). Moreover, if P2 is an antibody against P1, the identity of the protein P1 can be verified.
Finally, the supershift assay can also be used to assess the stoichiometry of a complex. For example, the ability of a protein to
multimerize on a nucleic acid substrate can be tested by varying
the length of the nucleic acid or the protein concentration.
1. Carry out a titration of P1 against the nucleic acid, as in
Subheading 3.5 above. Select a concentration of P1 where
the free nucleic acid band has completely disappeared.
2. Keeping the concentration of P1 constant, titrate an increasing
concentration of the putative binding protein P2 against the
protein–nucleic acid complex. It is important to keep one
sample with no protein as the negative control.
EMSAs to Detect Protein-DNA/RNA Interactions
329