dissociate during electrophoresis. This will depend on the particular complex under study since there are examples of EMSA
experiments that agree well with both equilibrium binding
experiments and kinetic measurements. Thus, a true equilibrium binding experiment or kinetic measurements should be
used in tandem with EMSA analysis.
33. The estimation of K d requires the protein concentration added
to the reaction (P tot ) to be approximately equal to the free
protein concentration after equilibrium is reached (P free ). If
the nucleic acid concentration is close to the K d of the interaction, depletion of the protein will underestimate the resulting
affinity. For example, if the K d is 100 nM and the same concentration of nucleic acid probe is added to the reaction, any
protein added to the reaction at and above this concentration
will begin to associate, deplete the “free” protein concentration and invalidate the assumption required for K d
estimation [19].
34. Image contrast and brightness adjustments must only be made
linearly and applied to an entire image or plate. Nonlinear
adjustments are strongly discouraged and are unsuitable for
semiquantitative analysis of protein–nucleic acid interactions,
as the measured intensity is no longer proportional to the
molarity of nucleic acid.
35. There are multiple ways of fitting the data. If the data are not
plotted logarithmically, they can be fitted with a single-site
binding equation (a form of the Langmuir isotherm) or a
quadratic binding function. If plotted logarithmically, the
data can also be fitted with a four-parameter logistic function.
All of these equations will contain terms that correspond to the
dissociation constant, where binding is half maximal.
36. To test whether P2, a protein known to bind to the P1–N1
protein–nucleic acid complex, competes with P1 binding to
nucleic acid N1, 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.
Titrate P2 against fixed concentrations of P1 and N1. A lack of
supershift indicates that P2 cannot bind simultaneously to a
P1–N1 complex. If the band corresponding to free N1 appears,
then P2 interaction with P1 precludes P1 binding to N1. If a
small shift in electrophoretic mobility occurs, then it may
indicate that the P2–N1 interaction is mutually exclusive with
the P1–N1 interaction.
37. Because two different nucleic acids are used in the assay, they
must be differentially labeled. For example, the two nucleic
acids can be labeled with different fluorophores with nonoverlapping excitation and emission spectra. Theoretically, this
EMSAs to Detect Protein-DNA/RNA Interactions
337
experiments that agree well with both equilibrium binding
experiments and kinetic measurements. Thus, a true equilibrium binding experiment or kinetic measurements should be
used in tandem with EMSA analysis.
33. The estimation of K d requires the protein concentration added
to the reaction (P tot ) to be approximately equal to the free
protein concentration after equilibrium is reached (P free ). If
the nucleic acid concentration is close to the K d of the interaction, depletion of the protein will underestimate the resulting
affinity. For example, if the K d is 100 nM and the same concentration of nucleic acid probe is added to the reaction, any
protein added to the reaction at and above this concentration
will begin to associate, deplete the “free” protein concentration and invalidate the assumption required for K d
estimation [19].
34. Image contrast and brightness adjustments must only be made
linearly and applied to an entire image or plate. Nonlinear
adjustments are strongly discouraged and are unsuitable for
semiquantitative analysis of protein–nucleic acid interactions,
as the measured intensity is no longer proportional to the
molarity of nucleic acid.
35. There are multiple ways of fitting the data. If the data are not
plotted logarithmically, they can be fitted with a single-site
binding equation (a form of the Langmuir isotherm) or a
quadratic binding function. If plotted logarithmically, the
data can also be fitted with a four-parameter logistic function.
All of these equations will contain terms that correspond to the
dissociation constant, where binding is half maximal.
36. To test whether P2, a protein known to bind to the P1–N1
protein–nucleic acid complex, competes with P1 binding to
nucleic acid N1, 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.
Titrate P2 against fixed concentrations of P1 and N1. A lack of
supershift indicates that P2 cannot bind simultaneously to a
P1–N1 complex. If the band corresponding to free N1 appears,
then P2 interaction with P1 precludes P1 binding to N1. If a
small shift in electrophoretic mobility occurs, then it may
indicate that the P2–N1 interaction is mutually exclusive with
the P1–N1 interaction.
37. Because two different nucleic acids are used in the assay, they
must be differentially labeled. For example, the two nucleic
acids can be labeled with different fluorophores with nonoverlapping excitation and emission spectra. Theoretically, this
EMSAs to Detect Protein-DNA/RNA Interactions
337