Alternatively, if the stoichiometry of only one protein
against the nucleic acid substrate is tested, titrate the protein
directly against the nucleic acid. Multimerization becomes visible at higher protein concentrations (Fig. 1b).
3. Carry out the EMSA and image the gel as described in Subheadings 3.1–3.4.
3.7 Variation:
Dual-Color
Competition EMSA
EMSAs can be carried out under competitive conditions with more
than one type of nucleic acid. The additional nucleic acid (N2) can
be unlabeled, or labeled differently from N1 (“two-color”) and can
be used to assess the nucleic acid binding specificity of the protein
(see Note 37). In this protocol, we detail a method for a dual-color
RNA EMSA [17].
1. This method requires the nucleic acids to be differentially
labeled (see Note 37). We routinely synthesize one substrate
with a 5
0 -FAM “blue” label and the other with a 5
0 -Alexa
647 “red” label.
2. The protein is titrated against fixed concentrations of two
nucleic acids (N1 and N2) (see Note 38). Perform a series of
protein dilutions to sample a particular range of concentrations. In the experiment shown in Fig. 2, a range from
50 nM to 2 μM was used.
3. Prepare a master mix solution of two different RNA concentrations, for example, 10 nM and 100 nM each RNA (20 nM and
200 nM total RNA) (see Note 39).
4. Assemble 10 μl binding reactions with the protein dilutions and
master mix (see Subheading 3.2). Incubate the mixtures for at
least 1 h to reach equilibrium.
5. Perform the EMSA as before (see Subheading 3.3).
6. Scan the gel (see Subheading 3.4) using the two nonoverlapping excitation wavelengths and emission filters (Typhoon FLA
Scanner, GE). Save each channel as a separate image file.
7. Using Adobe Photoshop or another suitable image-processing
software, convert each image to 8-bit grayscale and set false
color either using the duotone mode or the channel mixer in
RGB. Ensure the resulting image is an RGB image and overlay
as a separate, partially transparent layer on the other false color
image (Fig. 2).
4 Notes
1. General-use laboratory benches (such as those used for plasmid
and protein purifications) should be cleaned using 70% (v/v)
ethanol followed by an RNase inactivating solution such as
RNaseZAP™ (Invitrogen) to reduce the risk of RNase or
330
James A. W. Stowell et al.
against the nucleic acid substrate is tested, titrate the protein
directly against the nucleic acid. Multimerization becomes visible at higher protein concentrations (Fig. 1b).
3. Carry out the EMSA and image the gel as described in Subheadings 3.1–3.4.
3.7 Variation:
Dual-Color
Competition EMSA
EMSAs can be carried out under competitive conditions with more
than one type of nucleic acid. The additional nucleic acid (N2) can
be unlabeled, or labeled differently from N1 (“two-color”) and can
be used to assess the nucleic acid binding specificity of the protein
(see Note 37). In this protocol, we detail a method for a dual-color
RNA EMSA [17].
1. This method requires the nucleic acids to be differentially
labeled (see Note 37). We routinely synthesize one substrate
with a 5
0 -FAM “blue” label and the other with a 5
0 -Alexa
647 “red” label.
2. The protein is titrated against fixed concentrations of two
nucleic acids (N1 and N2) (see Note 38). Perform a series of
protein dilutions to sample a particular range of concentrations. In the experiment shown in Fig. 2, a range from
50 nM to 2 μM was used.
3. Prepare a master mix solution of two different RNA concentrations, for example, 10 nM and 100 nM each RNA (20 nM and
200 nM total RNA) (see Note 39).
4. Assemble 10 μl binding reactions with the protein dilutions and
master mix (see Subheading 3.2). Incubate the mixtures for at
least 1 h to reach equilibrium.
5. Perform the EMSA as before (see Subheading 3.3).
6. Scan the gel (see Subheading 3.4) using the two nonoverlapping excitation wavelengths and emission filters (Typhoon FLA
Scanner, GE). Save each channel as a separate image file.
7. Using Adobe Photoshop or another suitable image-processing
software, convert each image to 8-bit grayscale and set false
color either using the duotone mode or the channel mixer in
RGB. Ensure the resulting image is an RGB image and overlay
as a separate, partially transparent layer on the other false color
image (Fig. 2).
4 Notes
1. General-use laboratory benches (such as those used for plasmid
and protein purifications) should be cleaned using 70% (v/v)
ethanol followed by an RNase inactivating solution such as
RNaseZAP™ (Invitrogen) to reduce the risk of RNase or
330
James A. W. Stowell et al.