EMSA was carried out as previously described (Kim et al., 2014 (link)), with minor modifications. Briefly, the indicated amount of purified Chd4 protein was incubated with 5 nM of biotinylated RNAs in binding buffer (50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 10 mM β-mercaptoethanol, 5% glycerol) for 40 min at 25°C. Bound protein-RNA complexes were resolved on 5% native polyacrylamide mini gels at 70V and 4°C for 2 h. The complexes were transferred to an Amersham Hybond-N+ nylon membrane (RPN303B; GE Healthcare), UV-crosslinked, and visualized using a Chemiluminescent Nucleic Acid Detection Module kit (89880; Thermo Fisher Scientific) according to the manufacturer’s manual.
Protocol detail
Chd4 Protein-RNA Binding Analysis
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Buffer
Chd4
Emsa
Glycerol
Membrane
Mercaptoethanol
Nacl
Nucleic acid
Nylon
Polyacrylamide gels
Protein
Tris
Corresponding Organization :
Other organizations : Korea Advanced Institute of Science and Technology, Institute for Basic Science, Pohang University of Science and Technology
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Variable analysis
independent variables
- Indicated amount of purified Chd4 protein
- Binding of Chd4 protein to biotinylated RNAs
- Binding buffer (50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 10 mM β-mercaptoethanol, 5% glycerol)
- Incubation time (40 min)
- Incubation temperature (25°C)
- Native polyacrylamide gel electrophoresis (5% gel, 70V, 4°C, 2 h)
- Transfer to nylon membrane and UV-crosslinking
- Not specified
- Not specified
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