RNA folding and pull-down assays were performed as described with modifications.35 (link) Briefly, synthetic 5′-biotinylated rG4 (Bio-rG4) wild type (WT) or mutant (MT) RNA oligonucleotides (Millipore Sigma) were diluted to 5 mM in folding buffer, heated to 95°C and cooled to 25°C. G-quadruplex formation determined by circular dichroism (CD). Whole cell extracts from HepAD38, Huh7 and HepaRG cells were incubated with folded biotinylated RNAs (online supplemental table S3 ), followed by pull down with streptavidin beads (Promega). Bound proteins analysed by immunoblotting.
Streptavidin beads
Manufactured by Promega
Sourced in United States
Streptavidin beads are a type of laboratory equipment used for biomolecular purification and detection. They are composed of streptavidin, a protein derived from the bacterium Streptomyces, which has a high affinity for the small molecule biotin. These beads can be used to capture and isolate biotinylated molecules, such as proteins, nucleic acids, or other biomolecules, from complex mixtures.
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Lab products found in correlation
4 protocols using streptavidin beads
1
RNA G-quadruplex Interactome Profiling
2
Biotin-labeled RhoJ DNA Pulldown Assay
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Nuclear proteins (∼100 μg) were incubated with biotin-labeled RhoJ DNA probe at room temperature for 1 h in 1× binding buffer (20 mM HEPESpH7.9, 0.1 mM EDTA, 4% glycerol, and 2 mM DTT) supplemented with BSA (50 μg per reaction), poly-dIdC, and sonicated salmon sperm DNA (100 μg per reaction). DNA-protein complexes formed were then captured by incubating with the streptavidin beads (Promega) for 1 h at 4°C on a shaking platform. Ternary complex (biotin-labeled DNA-protein-streptavidin) was washed three times with 1× binding buffer supplemented with 0.01% Triton X and 100 mM KCl for 10 min each wash. The bound proteins were eluted with 1× SDS electrophoresis sample buffer by incubating at 90°C for 10 min and analyzed by SDS-PAGE gel electrophoresis followed by Western. Experiments were repeated at least three times.
3
RNA Pull-Down Assay for Interactome Analysis
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For the RNA pull-down assay, 100 pmol of RNA construct per reaction diluted in RNA-binding buffer [50 mM HEPES (pH 7.4), 100 mM KCl, 10 mM MgCl2, 0.5% Igepal CA-630] was incubated with 50 µL of streptavidin beads (Promega, Madison, Wisconsin, USA) for 30 min at room temperature (RT). RiboLock RNase inhibitors were used throughout the experiment (1:100). The supernatant was removed, and the beads were washed 2× with 500 μL of RNA-binding buffer; tRNA from baker’s yeast (R8508, Sigma Aldrich, St. Louis, Missouri, USA) was added to cytoplasmic and nuclear mouse tissue fractions and to lysates from human postmortem brain tissue. The beads with bound RNA were rotated with 1 mL of cytoplasmic or nuclear fraction for 4 h at 4 °C. The beads were washed 4× with 500 μL of RNA-binding buffer. Elution was performed with 3U RNase I (EN0601, Thermo Fisher Scientific, Waltham, Massachusetts, USA) for 10 min at 37 °C. Cytoplasmic and nuclear mouse tissue fractions (two independent experimental repeats), lysates from human postmortem brain tissue (two independent experimental repeats), HEK293 (three independent experimental repeats) and HEK293T (two independent experimental repeats) cell lysate were used for RNA pull-down assay.
4
Transcription Factor Binding Assay
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DNA affinity pull-down assay was performed as previously described (Chen et al., 2020b (link)). Nuclear proteins (~100 μg) were incubated with biotin-labeled RhoJ DNA probe at room temperature for 1 h in 1X binding buffer (20 mM HEPESpH7.9, 0.1 mM EDTA, 4% glycerol, 2 mM DTT) supplemented with BSA (50 μg per reaction), poly-dIdC, and sonicated salmon sperm DNA (100 μg per reaction). DNA-protein complexes formed were then captured by incubating with the streptavidin beads (Promega) for 1 h at 4°C on a shaking platform. Ternary complex (biotin-labeled DNA-protein-streptavidin) was washed three times with 1X binding buffer supplemented with 0.01% Triton X and 100 mM KCl for 10 min each wash. Eluates from the DNA affinity pull-down experiments were used for in vitro HDM assay using a commercially available kit (EpiQuik, Epigetek) per vendor recommendations. The assay was performed in triplicate wells and repeated three times. Data were normalized to the control group and expressed as relative HDM activity.
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