24. The time required to reach equilibrium will depend on the
nature of the protein–nucleic acid interaction and the reaction
conditions. Failing to attain equilibrium may lead to misleading results and failure to reproduce observations. At high
protein concentrations, the observed rate constant is dominated by the on-rate (k on ) [19]. In practice, k on is often 10
5
to 10
6 M
À1 s
À1 . At low protein concentrations, however, the
observed rate constant is instead dominated by the off-rate
(k off ) [19]. For a high affinity interaction with a K d of
1–10 nM, k off can be in the range of 0.001 to 0.0001 s
À1 and
the complex has a half-life (t 1/2 ) of ~10 min to >100 min.
Since the time taken to reach close to equilibrium is 5 Â t 1/2 , it
can thus take hours to reach equilibrium with low protein
concentrations. For an interaction with a binding affinity of
100 nM, the complex has a t 1/2 of 1–10 min and equilibrium
should be obtained in 5–50 min. It is thus safe to assume that
for interactions with affinities in this region, equilibrium will be
reached after a 60-min incubation. Also see Note 32.
25. Running the gel at low temperatures can be beneficial for two
reasons. Firstly, heat dissipates more evenly from the gel into
the surrounding cold buffer, ameliorating localized heating
which causes uneven bands. Secondly, since the dissociation
rate of the interaction is a function of temperature, running the
gel at ~4
C will increase the t 1/2 for the interaction and
potentially lead to a better estimate of the K d (see Notes 24
and 32).
26. We do not observe major differences between pre-run PAGE
gels and loading the samples directly. However, pre-running
the gel can remove excess ammonium and persulfate ions and
other impurities, such as acrylic acid, that could interfere with
complex formation and gel running.
27. We prefer to use tapered “gel-loading” tips to ensure that the
sample is evenly distributed along the bottom of the well. Small
sample volumes are advantageous as they result in sharper
bands at the end of electrophoresis, but the bottom of the
well must be evenly covered. For typical gels, this is approximately 5 μl.
28. While the nucleic acid alone will likely move through the
polyacrylamide matrix, the mobility of protein (or protein–
nucleic acid complex) through the gel will primarily depend
on two factors. Firstly, the molecular weight (and shape) of the
protein affects how the complex migrates through the gel. The
larger the protein, the slower it will move through the polyacrylamide matrix. Secondly, because the assay is carried out
near physiological pH, the isoelectric point (pI) and thus net
charge of the protein will also affect its mobility. Only proteins
EMSAs to Detect Protein-DNA/RNA Interactions
335
nature of the protein–nucleic acid interaction and the reaction
conditions. Failing to attain equilibrium may lead to misleading results and failure to reproduce observations. At high
protein concentrations, the observed rate constant is dominated by the on-rate (k on ) [19]. In practice, k on is often 10
5
to 10
6 M
À1 s
À1 . At low protein concentrations, however, the
observed rate constant is instead dominated by the off-rate
(k off ) [19]. For a high affinity interaction with a K d of
1–10 nM, k off can be in the range of 0.001 to 0.0001 s
À1 and
the complex has a half-life (t 1/2 ) of ~10 min to >100 min.
Since the time taken to reach close to equilibrium is 5 Â t 1/2 , it
can thus take hours to reach equilibrium with low protein
concentrations. For an interaction with a binding affinity of
100 nM, the complex has a t 1/2 of 1–10 min and equilibrium
should be obtained in 5–50 min. It is thus safe to assume that
for interactions with affinities in this region, equilibrium will be
reached after a 60-min incubation. Also see Note 32.
25. Running the gel at low temperatures can be beneficial for two
reasons. Firstly, heat dissipates more evenly from the gel into
the surrounding cold buffer, ameliorating localized heating
which causes uneven bands. Secondly, since the dissociation
rate of the interaction is a function of temperature, running the
gel at ~4
C will increase the t 1/2 for the interaction and
potentially lead to a better estimate of the K d (see Notes 24
and 32).
26. We do not observe major differences between pre-run PAGE
gels and loading the samples directly. However, pre-running
the gel can remove excess ammonium and persulfate ions and
other impurities, such as acrylic acid, that could interfere with
complex formation and gel running.
27. We prefer to use tapered “gel-loading” tips to ensure that the
sample is evenly distributed along the bottom of the well. Small
sample volumes are advantageous as they result in sharper
bands at the end of electrophoresis, but the bottom of the
well must be evenly covered. For typical gels, this is approximately 5 μl.
28. While the nucleic acid alone will likely move through the
polyacrylamide matrix, the mobility of protein (or protein–
nucleic acid complex) through the gel will primarily depend
on two factors. Firstly, the molecular weight (and shape) of the
protein affects how the complex migrates through the gel. The
larger the protein, the slower it will move through the polyacrylamide matrix. Secondly, because the assay is carried out
near physiological pH, the isoelectric point (pI) and thus net
charge of the protein will also affect its mobility. Only proteins
EMSAs to Detect Protein-DNA/RNA Interactions
335