5. If using fluorescence stains: Dissolve or dilute the stain in 1Â
TBE according to manufacturers’ instructions. For SYBR
Green II (Invitrogen), for each gel, 2 μl of the stain is diluted
10,000Â in 20 ml 1Â TBE buffer. Carefully transfer each gel to
a separate clean and opaque container containing the stain, and
proceed with staining and destaining according to manufacturer’s instructions. For SYBR Green II, the gel is transferred
to the diluted stain in a clean plastic container and allowed to
stain for 10 min on a benchtop orbital shaker. The gel is then
washed in 1Â TBE for 10 min.
6. Carefully transfer the gel to an appropriate imaging stage and
proceed with imaging (see Note 31) (Fig. 1b). If fluorescent
detection methods are used, an appropriate combination of
stimulation lasers and emission filters should be selected. For
SYBR Safe and 6-FAM, we use an excitation wavelength of
473 nm and a filter of 510 nm for detection of fluorescence
emission.
7. If protein staining is required after nucleic acid imaging, the gel
can be carefully removed and stained by a protein-specific stain
such as Coomassie blue according to manufacturers’ instructions. An image of the protein in the gel can be overlaid on the
previously obtained image of nucleic acid in image analysis
software. If the protein band and shifted nucleic acid band
coincide, then the shifted band likely corresponds to a protein–nucleic acid complex.
3.5 Variation:
Semiquantitative
Estimation
of Interaction Affinity
By titrating the protein against a fixed and low concentration of
nucleic acid, the affinity of the protein–nucleic acid interaction can
be estimated. This is carried out using densitometry of the band
corresponding to free nucleic acid. The estimate is valid only if the
method of detection is proportional to nucleic acid concentration.
It must be noted that the observed dissociation constant is only an
estimate as the measurement is not carried out under true solution
equilibrium conditions (see Note 32). We therefore normally use
EMSAs to qualitatively assay the differences between different protein constructs/point mutants and nucleic acid substrates.
1. Prepare a twofold dilution series of protein in sample buffer;
the maximum concentration of the protein should ideally be
~100Â greater than the expected dissociation constant of the
interaction. To avoid pipetting errors, choose a suitable volume
(~50 μl). Prepare the highest protein concentration in dilution
buffer in double this volume (i.e., 100 μl). Prepare a series of
tubes with 50 μl of dilution buffer. Serially dilute the protein by
taking 50 μl from the tube with the higher concentration,
mixing by pipetting up and down thoroughly, and drawing
up 50 μl to dilute in the next tube.
328
James A. W. Stowell et al.
TBE according to manufacturers’ instructions. For SYBR
Green II (Invitrogen), for each gel, 2 μl of the stain is diluted
10,000Â in 20 ml 1Â TBE buffer. Carefully transfer each gel to
a separate clean and opaque container containing the stain, and
proceed with staining and destaining according to manufacturer’s instructions. For SYBR Green II, the gel is transferred
to the diluted stain in a clean plastic container and allowed to
stain for 10 min on a benchtop orbital shaker. The gel is then
washed in 1Â TBE for 10 min.
6. Carefully transfer the gel to an appropriate imaging stage and
proceed with imaging (see Note 31) (Fig. 1b). If fluorescent
detection methods are used, an appropriate combination of
stimulation lasers and emission filters should be selected. For
SYBR Safe and 6-FAM, we use an excitation wavelength of
473 nm and a filter of 510 nm for detection of fluorescence
emission.
7. If protein staining is required after nucleic acid imaging, the gel
can be carefully removed and stained by a protein-specific stain
such as Coomassie blue according to manufacturers’ instructions. An image of the protein in the gel can be overlaid on the
previously obtained image of nucleic acid in image analysis
software. If the protein band and shifted nucleic acid band
coincide, then the shifted band likely corresponds to a protein–nucleic acid complex.
3.5 Variation:
Semiquantitative
Estimation
of Interaction Affinity
By titrating the protein against a fixed and low concentration of
nucleic acid, the affinity of the protein–nucleic acid interaction can
be estimated. This is carried out using densitometry of the band
corresponding to free nucleic acid. The estimate is valid only if the
method of detection is proportional to nucleic acid concentration.
It must be noted that the observed dissociation constant is only an
estimate as the measurement is not carried out under true solution
equilibrium conditions (see Note 32). We therefore normally use
EMSAs to qualitatively assay the differences between different protein constructs/point mutants and nucleic acid substrates.
1. Prepare a twofold dilution series of protein in sample buffer;
the maximum concentration of the protein should ideally be
~100Â greater than the expected dissociation constant of the
interaction. To avoid pipetting errors, choose a suitable volume
(~50 μl). Prepare the highest protein concentration in dilution
buffer in double this volume (i.e., 100 μl). Prepare a series of
tubes with 50 μl of dilution buffer. Serially dilute the protein by
taking 50 μl from the tube with the higher concentration,
mixing by pipetting up and down thoroughly, and drawing
up 50 μl to dilute in the next tube.
328
James A. W. Stowell et al.