The electrophoresis mobility shift assay of recombinant TRBP protein was described previously (41 (link)). Briefly, the recombinant TRBP protein was incubated with 32P-labeled miLuc-1 or miLuc-2 for 30 min on ice in EMSA binding buffer containing 20 mM Tris–HCl [pH8.0], 1.5 mM MgCl2, 50 mM NaCl, 1.5 mM DTT, 100 ng/μl sonicated salmon sperm DNA, 5% glycerol and 0.4 U/ml RNasein (Promega). The samples were electrophoresed on a 9% non-denatured polyacrylamide gel in 0.25× TBE buffer. The visualization was performed using the Fuji imaging plate and Typhoon image analyzer (GE Healthcare).
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TRBP Binding to miRNA by EMSA
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Corresponding Organization : The University of Tokyo
Other organizations : Chiba University, Kyoto University, Maebashi Institute of Technology
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Variable analysis
independent variables
- Incubation of recombinant TRBP protein with 32P-labeled miLuc-1 or miLuc-2
- Electrophoretic mobility shift of the TRBP-miRNA complexes
- EMSA binding buffer containing 20 mM Tris–HCl [pH8.0], 1.5 mM MgCl2, 50 mM NaCl, 1.5 mM DTT, 100 ng/μl sonicated salmon sperm DNA, 5% glycerol and 0.4 U/ml RNasein (Promega)
- Electrophoresis on a 9% non-denatured polyacrylamide gel in 0.25× TBE buffer
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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