may be helpful to silanize the glass surface to allow easier
removal of gels from the plates. To do so, wipe the silanization
solution with lint-free paper over clean and dry glass plates and
leave the solution to dry in a fume hood (see Note 7).
2. Assemble the gel caster according to manufacturer’s specifications (see Notes 6 and 14).
3. For a mini-gel, add 35 μl 10% APS and 3.5 μl TEMED to 7 ml
of native polyacrylamide gel mix (see Note 3) in a sterile 15 ml
conical bottom centrifuge tube. Mix well by gentle inversion.
For more than one gel or larger gels, reagent volumes can be
scaled up correspondingly.
4. Slowly pour the resulting mixture into the assembled
gel-casting apparatus. Immediately insert a comb between the
glass plates, taking care not to introduce any air bubbles (see
Notes 15 and 16).
5. Leave the gels to polymerize at room temperature for at least
2 h and at most overnight (see Note 17). Once polymerized,
gels can be kept up for up to 1 week at 4
C covered with paper
towels soaked in 1Â TBE buffer and sealed in plastic wrap.
3.2 Sample
Preparation
1. In a typical experiment, the concentration of nucleic acid is
kept constant and the concentration of protein is varied (see
Notes 18 and 19). It is important to include a negative control
with no protein. A positive control with a protein which is
known to bind the nucleic acid can also be included.
2. Prepare 10Â protein stocks in a dilution buffer (see Notes
8 and 20–22) in 1.5 ml microcentrifuge tubes. This can be a
dilution series (see Subheading 3.5) or a smaller range of concentrations (see Subheadings 3.6 and 3.7).
3. Prepare a master mix of nucleic acid, EMSA buffer, and loading
dye in a 1.5 ml microcentrifuge tube (see Note 23). For example, for nine 10 μl binding reactions, assemble a master mix for
10 reactions (to account for dead volume when pipetting) as
follows: 10 μl 10Â RNA stock, 10 μl 10Â EMSA buffer, and
10 μl loading dye in 90 μl total volume. This can be scaled
accordingly.
4. In 0.2 or 0.5 ml tubes, pipette 1 μl of the 10Â protein stock,
followed by 9 μl of the master mix. Mix by gently pipetting up
and down.
5. Incubate the sample at room temperature for at least 1 h to
allow the interaction to reach equilibrium (see Note 24).
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James A. W. Stowell et al.
removal of gels from the plates. To do so, wipe the silanization
solution with lint-free paper over clean and dry glass plates and
leave the solution to dry in a fume hood (see Note 7).
2. Assemble the gel caster according to manufacturer’s specifications (see Notes 6 and 14).
3. For a mini-gel, add 35 μl 10% APS and 3.5 μl TEMED to 7 ml
of native polyacrylamide gel mix (see Note 3) in a sterile 15 ml
conical bottom centrifuge tube. Mix well by gentle inversion.
For more than one gel or larger gels, reagent volumes can be
scaled up correspondingly.
4. Slowly pour the resulting mixture into the assembled
gel-casting apparatus. Immediately insert a comb between the
glass plates, taking care not to introduce any air bubbles (see
Notes 15 and 16).
5. Leave the gels to polymerize at room temperature for at least
2 h and at most overnight (see Note 17). Once polymerized,
gels can be kept up for up to 1 week at 4
C covered with paper
towels soaked in 1Â TBE buffer and sealed in plastic wrap.
3.2 Sample
Preparation
1. In a typical experiment, the concentration of nucleic acid is
kept constant and the concentration of protein is varied (see
Notes 18 and 19). It is important to include a negative control
with no protein. A positive control with a protein which is
known to bind the nucleic acid can also be included.
2. Prepare 10Â protein stocks in a dilution buffer (see Notes
8 and 20–22) in 1.5 ml microcentrifuge tubes. This can be a
dilution series (see Subheading 3.5) or a smaller range of concentrations (see Subheadings 3.6 and 3.7).
3. Prepare a master mix of nucleic acid, EMSA buffer, and loading
dye in a 1.5 ml microcentrifuge tube (see Note 23). For example, for nine 10 μl binding reactions, assemble a master mix for
10 reactions (to account for dead volume when pipetting) as
follows: 10 μl 10Â RNA stock, 10 μl 10Â EMSA buffer, and
10 μl loading dye in 90 μl total volume. This can be scaled
accordingly.
4. In 0.2 or 0.5 ml tubes, pipette 1 μl of the 10Â protein stock,
followed by 9 μl of the master mix. Mix by gently pipetting up
and down.
5. Incubate the sample at room temperature for at least 1 h to
allow the interaction to reach equilibrium (see Note 24).
326
James A. W. Stowell et al.