Binding reactions (20 μl) were carried out in 25 mM Tris-HCl pH 7.5, 15% glycerol, 100 mM NaCl, 2 mM DTT, 5 mM EDTA and 1 mg/ml BSA with 1 nM DNA substrate and the indicated concentrations of MutLγ. Complexes were assembled for 30 minutes at 30°C and separated on a 5% Tris-acetate-EDTA-polyacrylamide/bis (80/1) gel. Gels were dried and radioactivity was detected by phosphorimaging (Fuji).
Phosphorimaging
Manufactured by Fujifilm
Sourced in Japan
Phosphorimaging is a laboratory equipment used for the detection and analysis of radioactive signals. It utilizes a phosphor screen to capture and store the energy from radioactive samples, which can then be scanned and analyzed using specialized software.
Automatically generated - may contain errors
6 protocols using phosphorimaging
2
Protein-DNA Binding Assay
3
Radioactive tRNA Binding Assay
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Lyophilized E. coli MRE 600 tRNA (Roche, Germany) was dissolved in ultrapure MilliQ water treated with diethylpyrocarbonate (DEPC). The 5′-ends of the tRNA molecules were labeled with [γ-32P]-ATP using T4 polynucleotide kinase (Thermo Scientific, Lithuania). The 5′-labeled RNAs were incubated with target proteins for 30 min at 37 °C in a binding mixture, containing 20 mM Tris-HCl (pH 7.5), 300 mM KCl, 10 mM MgCl2 or 10 mM CoCl2, and 100 ng/µL bovine serum albumin, similarly as described by Kim et al. [5 (link)]. The final ratio of tRNA to protein was 1:100. After the incubation, samples were loaded onto 8% (v/v) native polyacrylamide gel. Electrophoresis was conducted at 150 V for 150 min at 1× TBE buffer. The visualization was performed by phosphorimaging (Fuji, Japan).
4
Protein-DNA Binding Assay
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Binding reactions (20 μl) were carried out in 25 mM Tris-HCl pH 7.5, 7.5% glycerol, 100 mM NaCl, 2 mM DTT, 5 mM MgCl2 and 1 mg/ml BSA with 0.1 nM DNA (for hairpin substrates) or 1 nM DNA (for mini-circles) and the indicated amounts of protein complexes. Complexes were assembled for 30 minutes at 30 °C and separated on a 5% Tris-acetate-polyacrylamide/bis (80:1) gel containing 0.5 mM MgCl2 at 200 V for two hours. Gels were dried then analyzed by phosphorimaging (Fuji). Apparent dissociation constants (KD) were estimated from protein titration EMSA experiments as the concentration of protein at which 50% of the substrate was bound. Because binding to 5′ overhang substrates was so tight (apparent KD close to the DNA substrate concentration in the assay), these measurements are approximations of the true KD.
5
Detecting Ubiquitin Conjugates by IMAC and In Vitro Ubiquitination Assay
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Cells were lysed in buffer containing 6 M guanidinium–HCl and protein–ubiquitin conjugates were captured by immobilized metal affinity chromatography (IMAC) on Nickel-Agarose beads (Qiagen) and washed in 8 M urea. After release from the beads conjugates were resolved by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by immunoblotting. The antibodies used are listed in Supplementary Experimental Procedures .
In vitro ubiquitination assays were performed as described elsewhere (33 (link)). Briefly, 35S-labelled ELK-1 was generated by cell-free expression using the coupled TNT reticulocyte lysate system (Promega). After removal of unincorporated 35S-methionine by gel filtration (Micro-Spin, BioRad), radio-labelled ELK-1 was incubated with UBQ (10 μg), rE1 (500 ng), rE2 (UBCH5; 500 ng), ATP (4 mM), DTT (1 mM), ubiquitin aldehyde (5 μM) and HeLa Nxt (15–30 μg) for 1 h at 30°C. Reactions were resolved on 8% SDS-PAGE gels, dried and visualized by phosphor-imaging (Fujifilm).
In vitro ubiquitination assays were performed as described elsewhere (33 (link)). Briefly, 35S-labelled ELK-1 was generated by cell-free expression using the coupled TNT reticulocyte lysate system (Promega). After removal of unincorporated 35S-methionine by gel filtration (Micro-Spin, BioRad), radio-labelled ELK-1 was incubated with UBQ (10 μg), rE1 (500 ng), rE2 (UBCH5; 500 ng), ATP (4 mM), DTT (1 mM), ubiquitin aldehyde (5 μM) and HeLa Nxt (15–30 μg) for 1 h at 30°C. Reactions were resolved on 8% SDS-PAGE gels, dried and visualized by phosphor-imaging (Fujifilm).
6
In vitro Processing of pre-rRNA
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Processing of pre-rRNA in vitro was performed as follows5 (link). Extract proteins (60–80 μg) from L1210 cells were incubated with 20 fmoles of 32P-labelled synthetic transcripts in 20 mM Hepes pH 7.9, 2 mM MgCl2, 0.14 mM EDTA, 1.5 mM ATP, 120 mM KCl, 2 mM DTT and 9% (v/v) glycerol. The reaction was terminated by the addition of an equal volume of 2% SDS, 12.5 mM EDTA, 0.4 mg ml−1 glycogen and 0.4 mg ml−1 proteinase K. After incubation for 30 min at 42 °C, radiolabelled RNA was analysed by electrophoresis on 6% polyacrylamide/TBE gels and PhosphorImaging (Fujifilm).
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