hnRNP U RIP experiments were performed in nuclear extracts isolated from unstimulated A549 cells under native conditions. Assays were performed as previously described (Tsai et al., 2010 (link)). Nuclear extracts were immunoprecipitated with 2.5 μg hnRNP U (Abcam, clone 4D11, ab6106) and isotype-matched control IgG antibodies overnight. RNA-protein-antibody complexes were captured using Dynabead Protein A/G (Thermo Fisher Scientific). RNA was eluted by adding TRIzol directly to magnetic beads and isolated as per manufacturer’s instructions. cDNA was synthesized using TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech) and analyzed by qPCR. Results were normalized to input RNA and shown as fold-enrichment over control IgG RIP.
Dynabead protein a g
Manufactured by Thermo Fisher Scientific
Dynabeads Protein A/G are magnetic beads coated with either Protein A or Protein G. They are used for the rapid and efficient immunoprecipitation and purification of target proteins from complex samples.
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2 protocols using dynabead protein a g
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hnRNP U RNA Immunoprecipitation Protocol
2
RNA Immunoprecipitation Assay Protocol
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The RNA immunoprecipitation (RIP) assay was performed as previously described[31 (link),32 (link)]. With IgG antibodies as the isotype-matched control, nuclear extracts were immunoprecipitated with 2.5 mg hnRNP U (Abcam, clone 4D11, ab6106) overnight. RNA-protein-antibody complexes were captured using Dynabead Protein A/G (Thermo Fisher Scientific). RNA was eluted by adding TRIzol directly to magnetic beads and isolated according to the manufacturer’s instructions. cDNA was synthesized using TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech) and analyzed by qPCR. The results were normalized to input RNA and are shown as fold-enrichment over control IgG RIP.
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