should be highly purified. RNAs can be prepared either by
in vitro transcription of a DNA template [12] or by commercial
chemical synthesis. In vitro transcribed RNAs should be purified by denaturing polyacrylamide gel electrophoresis and by
gel extraction, either by crush and soak [13] or electroelution
[14] after excising the correct band.
5. 10Â EMSA buffer: 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5, 100 mM sodium
chloride (NaCl), 2 mM magnesium acetate (MgAc), 0.1 mM
tris(2-carboxyethyl)phosphine (TCEP). The composition of
the EMSA buffer will depend on the experiment and nature
of the interaction (see Note 8).
2.3 Detection
Methods
Additional materials depend on the method of detection.
1. Fluorescent stain: SYBR Safe (DNA, Thermo Fisher) or SYBR
Green II (RNA, Thermo Fisher) (see Note 10), visible with a
blue light or UV transilluminator.
2. Fluorescent label: Fluorescent labels can be incorporated during chemical synthesis of DNA or RNA. Alternatively, nucleic
acids can be fluorescently labeled with a homemade or commercially available kit (see Notes 11, and 12). An example is the
5
0 - or 3
0 -end EndTag labeling kit (Vector Laboratories) used
with a conjugable fluorophore (e.g., fluorescein maleimide,
Vector Laboratories catalog number SP-1502-12).
3. Radioactive label: γ32 P dNTP/γ32 P rATP for end labeling,
α32 P NTPs for internal labeling. Radioactive reagents should
be handled according to local safety procedures. The nucleic
acid can be radioactively end-labeled by a relatively inexpensive
protocol using T4 polynucleotide kinase [15].
4. Gel dryer (Bio-Rad) or similar platform.
5. Amersham Typhoon FLA Imager (GE) or similar platform
with multiple lasers/filters (see Note 13) if using fluorescence
or phosphorimaging and quantification.
6. Coomassie blue stain (or equivalent) if visualization of protein
bands is required.
3 Methods
Carry out all procedures at room temperature unless otherwise
specified.
3.1 Preparation
of Polyacrylamide Gel
1. Ensure that the glass plates and combs are clean. If not, clean,
rinse with 70% (v/v) ethanol and allow components to thoroughly dry before continuing. For larger gel-casting systems, it
EMSAs to Detect Protein-DNA/RNA Interactions
325
in vitro transcription of a DNA template [12] or by commercial
chemical synthesis. In vitro transcribed RNAs should be purified by denaturing polyacrylamide gel electrophoresis and by
gel extraction, either by crush and soak [13] or electroelution
[14] after excising the correct band.
5. 10Â EMSA buffer: 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.5, 100 mM sodium
chloride (NaCl), 2 mM magnesium acetate (MgAc), 0.1 mM
tris(2-carboxyethyl)phosphine (TCEP). The composition of
the EMSA buffer will depend on the experiment and nature
of the interaction (see Note 8).
2.3 Detection
Methods
Additional materials depend on the method of detection.
1. Fluorescent stain: SYBR Safe (DNA, Thermo Fisher) or SYBR
Green II (RNA, Thermo Fisher) (see Note 10), visible with a
blue light or UV transilluminator.
2. Fluorescent label: Fluorescent labels can be incorporated during chemical synthesis of DNA or RNA. Alternatively, nucleic
acids can be fluorescently labeled with a homemade or commercially available kit (see Notes 11, and 12). An example is the
5
0 - or 3
0 -end EndTag labeling kit (Vector Laboratories) used
with a conjugable fluorophore (e.g., fluorescein maleimide,
Vector Laboratories catalog number SP-1502-12).
3. Radioactive label: γ32 P dNTP/γ32 P rATP for end labeling,
α32 P NTPs for internal labeling. Radioactive reagents should
be handled according to local safety procedures. The nucleic
acid can be radioactively end-labeled by a relatively inexpensive
protocol using T4 polynucleotide kinase [15].
4. Gel dryer (Bio-Rad) or similar platform.
5. Amersham Typhoon FLA Imager (GE) or similar platform
with multiple lasers/filters (see Note 13) if using fluorescence
or phosphorimaging and quantification.
6. Coomassie blue stain (or equivalent) if visualization of protein
bands is required.
3 Methods
Carry out all procedures at room temperature unless otherwise
specified.
3.1 Preparation
of Polyacrylamide Gel
1. Ensure that the glass plates and combs are clean. If not, clean,
rinse with 70% (v/v) ethanol and allow components to thoroughly dry before continuing. For larger gel-casting systems, it
EMSAs to Detect Protein-DNA/RNA Interactions
325