3.3 Polyacrylamide
Gel Electrophoresis
1. Rinse the glass plates containing the completely polymerized
polyacrylamide gel with MilliQ water to remove gel debris.
2. If the gel is to be run at 4
C, prechill 1Â TBE buffer (see Note
25).
3. In a suitable electrophoretic chamber, assemble the gel plates
and add 1Â TBE buffer. The electrophoretic apparatus should
be filled such that the upper chamber is full and the bottom of
the glass plate is submerged in the bottom chamber. Reassemble the electrophoretic apparatus if any leaks occur at this stage.
Buffer in the bottom chamber can be filled to cover most of the
gel to act as a heat sink, minimizing gel heating. Remove the
gel comb and any debris from the wells.
4. If desired, the gel can be pre-run at 100 V for at least 30 min
(see Note 26).
5. Carefully add the samples to the center of the bottom of the
wells of the gel (see Note 27). The loaded tip should be near
the bottom of the well, and the sample should be slowly
expelled, without introducing any air bubbles.
6. Electrophorese the sample at 100 V at the desired temperature.
Ensure that the electrophoretic chamber and buffer do not
exceed the desired temperature. The negatively charged nucleic
acid and any stably bound proteins will move through the gel
towards the anode (see Note 28).
7. When the Orange G dye front reaches the bottom of the gel
plate (~60 min) (see Note 29), turn off the power supply.
3.4 Gel Imaging
1. Disassemble the gel apparatus. Carefully pry the gel plates apart
using either a metal spatula or a plastic wedge. The gel should
remain on one of the two glass plates.
2. Rinse the gel with deionized water.
3. How the gel is handled at this stage will depend on the detection method to be used (see Note 30). For colorimetric or
fluorescent stains, the gel should be stained. When the nucleic
acid has been directly labeled by fluorescent dyes or radioactive
isotopes, no staining is required. We use fluorescent labels or
fluorescent stains due to their reasonably high sensitivity.
4. If using radiolabeled nucleic acids: The resulting gels must be
exposed to a phosphorimaging screen. If the sample is sufficiently radioactive and the detection method sufficiently sensitive, the gel can be covered with plastic wrap and imaged
directly on the phosphorimaging screen. Alternatively, the gel
can be dried prior to imaging. To do so, the gel is sandwiched
between a sheet of filter paper and plastic wrap and dried in the
gel dryer, with the plastic wrap facing upwards. Drying should
be complete to avoid cracking of the gel. This process typically
takes 2 h to complete.
EMSAs to Detect Protein-DNA/RNA Interactions
327
Gel Electrophoresis
1. Rinse the glass plates containing the completely polymerized
polyacrylamide gel with MilliQ water to remove gel debris.
2. If the gel is to be run at 4
C, prechill 1Â TBE buffer (see Note
25).
3. In a suitable electrophoretic chamber, assemble the gel plates
and add 1Â TBE buffer. The electrophoretic apparatus should
be filled such that the upper chamber is full and the bottom of
the glass plate is submerged in the bottom chamber. Reassemble the electrophoretic apparatus if any leaks occur at this stage.
Buffer in the bottom chamber can be filled to cover most of the
gel to act as a heat sink, minimizing gel heating. Remove the
gel comb and any debris from the wells.
4. If desired, the gel can be pre-run at 100 V for at least 30 min
(see Note 26).
5. Carefully add the samples to the center of the bottom of the
wells of the gel (see Note 27). The loaded tip should be near
the bottom of the well, and the sample should be slowly
expelled, without introducing any air bubbles.
6. Electrophorese the sample at 100 V at the desired temperature.
Ensure that the electrophoretic chamber and buffer do not
exceed the desired temperature. The negatively charged nucleic
acid and any stably bound proteins will move through the gel
towards the anode (see Note 28).
7. When the Orange G dye front reaches the bottom of the gel
plate (~60 min) (see Note 29), turn off the power supply.
3.4 Gel Imaging
1. Disassemble the gel apparatus. Carefully pry the gel plates apart
using either a metal spatula or a plastic wedge. The gel should
remain on one of the two glass plates.
2. Rinse the gel with deionized water.
3. How the gel is handled at this stage will depend on the detection method to be used (see Note 30). For colorimetric or
fluorescent stains, the gel should be stained. When the nucleic
acid has been directly labeled by fluorescent dyes or radioactive
isotopes, no staining is required. We use fluorescent labels or
fluorescent stains due to their reasonably high sensitivity.
4. If using radiolabeled nucleic acids: The resulting gels must be
exposed to a phosphorimaging screen. If the sample is sufficiently radioactive and the detection method sufficiently sensitive, the gel can be covered with plastic wrap and imaged
directly on the phosphorimaging screen. Alternatively, the gel
can be dried prior to imaging. To do so, the gel is sandwiched
between a sheet of filter paper and plastic wrap and dried in the
gel dryer, with the plastic wrap facing upwards. Drying should
be complete to avoid cracking of the gel. This process typically
takes 2 h to complete.
EMSAs to Detect Protein-DNA/RNA Interactions
327