PMC:2728203 / 32685-33728 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2728203","sourcedb":"PMC","sourceid":"2728203","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2728203","text":"Gel shift RNA binding assay\nSynthetic 25-nt poly(N) RNAs (Dharmacon) were 5′-end labeled with [γ-32P]ATP (GE Healthcare Life Sciences) using T4 polynucleotide kinase (Promega). The reaction was stopped, and unincorporated nucleotides were removed using Qiagen's Nucleotide Removal Kit. GST, GST-Nab2-N, and, as a positive control, GST-Srm160-N35 were expressed in E. coli and purified using glutathione beads as described above for in vitro binding assays. RNA electrophoretic mobility shift assays were performed by incubating the increasing amounts (0.5–5 μM) of recombinant GST, GST-Nab2-N, or GST-Srm160-N with approximately 30 pM radioactively labeled poly(N) RNA oligonucleotide (a 25-mer RNA oligonucleotide with each position randomized) in binding buffer for 30 min at 20 °C. Binding reactions were loaded onto a 5% native polyacrylamide gel and electrophoresed at 30 mA in 0.3× TBE buffer for 30 min to separate free oligonucleotide from protein–RNA complexes. Gels were dried and exposed overnight using a phosphorimager (Amersham).","divisions":[{"label":"title","span":{"begin":0,"end":27}}],"tracks":[]}