Samples containing 5 nM labeled duplex RNA and 0 to 300 nM enzyme were incubated in 15 mM Tris-HCl pH 7.5, 26 mM KCl, 40 mM potassium glutamate, 1.5 mM EDTA, 0.003% (v/v) NP-40, 4% glycerol, 0.5 mM dithiothreitol (DTT), 1 μg/mL yeast tRNA, 0.16 U/μL RNAse inhibitor, and 0.2 mg/mL BSA at 30 °C for 30 min. Samples were loaded onto a 6% 80:1 acrylamide/bis-acrylamide gel and electrophoresed under non-denaturing conditions in 1X TBE at 4 °C for 1.5 h. The gels were dried under reduced pressure at 80 °C for 1.5 h, then exposed to storage phosphor imaging plates (Kodak) in the dark. Plates were scanned using a Typhoon imaging system (GE Healthcare) and band intensities were quantified by ImageQuant (GE Healthcare). Data were plotted (fraction RNA bound = protein-RNA / (protein-RNA + free RNA)) and analyzed using Microsoft Excel and GraphPad Prism. The band intensities arising from protein-RNA aggregation in the wells were excluded in final data processing as it does not appreciably change the KD values derived from the binding curves, whether included or excluded from the calculation for fraction of RNA bound to the protein.
Storage phosphor imaging plates
Manufactured by Kodak
Storage phosphor imaging plates are a type of lab equipment used for capturing and storing digital images. They function by absorbing and storing energy from radiation, which can then be read and converted into a digital image. These plates offer a high-resolution, cost-effective solution for various imaging applications in the scientific and medical fields.
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2 protocols using storage phosphor imaging plates
1
In Vitro RNA-Protein Binding Assay
2
Quantitative RNA Binding Analysis
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Oligonucleotides were 5′-end labeled using [γ-32P] ATP (6000 Ci/mmol) and T4 polynucleotide kinase, purified by 19% denaturing polyacrylamide gel in 1× TBE, visualized by storage phosphor autoradiography, excised, and extracted by crush and soak. The labeled oligonucleotide was hybridized to its unlabeled complement by heating to 95°C for 5 min in 1× TE and 200 mM NaCl, followed by slow cooling to room temperature. Samples containing less than 1.1 nM RNA and different concentrations of hADAR2-R2D mutants (64, 32, 16, 8, 4, 2, 1, 0.5, 0.25 and 0 nM) were equilibrated in 20 mM Tris–HCl, pH 7.0, 3.5% glycerol, 0.5% DTT, 60 mM KCl, 20 mM NaCl, 0.1 mM BME, 1.5 mM EDTA, 0.003% NP-40, 160 units/ml RNasin, 100 μg/ml BSA, and 1.0 μg/ml yeast tRNA for 30 min at 30°C. Samples were loaded onto a running 6% nondenaturing polyacrylamide gel (79:1 acrylamide:bisacrylamide) and electrophoresed in 1× TBE buffer at 4°C for 90 min. The gels were dried on a gel drier (Biorad) for 60 min at 80°C under vacuum followed by exposure to storage phosphor imaging plates (Kodak) for 3 h in the dark. After exposure, the dried polyacrylamide gels were removed, and the phosphor imaging plates were scanned by Typhoon Trio Variable Mode Imager (GE Healthcare). See Supplementary Table S2 for RNA sequences.
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