combined with other techniques such as Western blotting [10] and
high-throughput sequencing [11] to extract additional information. Nonetheless, EMSAs have some limitations as complex formation is not in true chemical equilibrium. Furthermore, EMSAs
do not provide information regarding the binding site on either the
protein or nucleic acid. Thus, observations from EMSAs are most
often verified using other complementary techniques, such as fluorescence polarization (Chapter 10), microscale thermophoresis
(Chapter 6), surface plasmon resonance (Chapter 17), or structural
methods.
Here, we outline the materials and steps required for EMSAs.
These include preparation of the native polyacrylamide gel, preparation of protein–nucleic acid samples, electrophoresis, and imaging. We further discuss several common adaptations of EMSAs,
including semiquantitative estimation of affinity, supershift assays,
and competitive EMSAs.
2 Materials
RNase and DNase contamination should be avoided. Therefore, it
is important to work on clean benches and to protect the samples
from contamination by wearing gloves at all times (see Note 1).
Furthermore, all solutions should be made up and diluted using
fresh ultrapure water (MilliQ) with analytical grade reagents. Solutions can also be treated with diethylpyrocarbonate (DEPC) and
autoclaved to avoid nuclease contamination (see Subheading 2.2).
Filter-sterilize all solutions with a 0.22 μm filter to remove precipitates or particulate contaminants. Take special precautions (according to local safety procedures) when handling toxic or radioactive
materials.
2.1 Native
Polyacrylamide Gel
1. 10Â TBE (Tris-Borate EDTA) stock: Weigh 108 g tris
(hydroxymethyl)aminomethane (Tris) base, 9.3 g ethylenediaminetetraacetic acid (EDTA), and 55 g boric acid. Dissolve in
water and top up to 1 l. Autoclave the resulting solution
(121
C, 15 min). The solution can be stored at room temperature indefinitely, but should be discarded if there is any visible
precipitation.
2. Isopropanol: Supplied at !99.9% purity (HPLC grade).
3. Native polyacrylamide gel mix: 8% gel stock (see Note 2). Mix
50 ml of 10Â TBE and 100 ml of 40% (w/v) 19:1 acrylamide:
bis-acrylamide solution (preferably gas-stabilized, see Note 3).
Make up to 500 ml with MilliQ water and degas by vacuum
filtration. This solution can be made fresh or stored as an
unpolymerized stock at 4
C for several months. We routinely
make an unpolymerized gel stock solution when performing
multiple experiments, using the desired volume when required.
EMSAs to Detect Protein-DNA/RNA Interactions
323
high-throughput sequencing [11] to extract additional information. Nonetheless, EMSAs have some limitations as complex formation is not in true chemical equilibrium. Furthermore, EMSAs
do not provide information regarding the binding site on either the
protein or nucleic acid. Thus, observations from EMSAs are most
often verified using other complementary techniques, such as fluorescence polarization (Chapter 10), microscale thermophoresis
(Chapter 6), surface plasmon resonance (Chapter 17), or structural
methods.
Here, we outline the materials and steps required for EMSAs.
These include preparation of the native polyacrylamide gel, preparation of protein–nucleic acid samples, electrophoresis, and imaging. We further discuss several common adaptations of EMSAs,
including semiquantitative estimation of affinity, supershift assays,
and competitive EMSAs.
2 Materials
RNase and DNase contamination should be avoided. Therefore, it
is important to work on clean benches and to protect the samples
from contamination by wearing gloves at all times (see Note 1).
Furthermore, all solutions should be made up and diluted using
fresh ultrapure water (MilliQ) with analytical grade reagents. Solutions can also be treated with diethylpyrocarbonate (DEPC) and
autoclaved to avoid nuclease contamination (see Subheading 2.2).
Filter-sterilize all solutions with a 0.22 μm filter to remove precipitates or particulate contaminants. Take special precautions (according to local safety procedures) when handling toxic or radioactive
materials.
2.1 Native
Polyacrylamide Gel
1. 10Â TBE (Tris-Borate EDTA) stock: Weigh 108 g tris
(hydroxymethyl)aminomethane (Tris) base, 9.3 g ethylenediaminetetraacetic acid (EDTA), and 55 g boric acid. Dissolve in
water and top up to 1 l. Autoclave the resulting solution
(121
C, 15 min). The solution can be stored at room temperature indefinitely, but should be discarded if there is any visible
precipitation.
2. Isopropanol: Supplied at !99.9% purity (HPLC grade).
3. Native polyacrylamide gel mix: 8% gel stock (see Note 2). Mix
50 ml of 10Â TBE and 100 ml of 40% (w/v) 19:1 acrylamide:
bis-acrylamide solution (preferably gas-stabilized, see Note 3).
Make up to 500 ml with MilliQ water and degas by vacuum
filtration. This solution can be made fresh or stored as an
unpolymerized stock at 4
C for several months. We routinely
make an unpolymerized gel stock solution when performing
multiple experiments, using the desired volume when required.
EMSAs to Detect Protein-DNA/RNA Interactions
323