Biotin-labeled RNA pull-down and western blot analysis
This protocol is extracted from research article:
Functional roles of antisense enhancer RNA for promoting prostate cancer progression
Theranostics, Jan 1, 2021; DOI: 10.7150/thno.51931

Biotin-labeled RNAs were in vitro transcript using Biotin RNA Labeling Mix (Roche) and T7 polymerase (New England Biolabs). C4-2 cells cultured in androgen-depleted medium were lysed in modified Binding buffer (150 mM NaCl, 50 mM Tris-HCl pH7.5, 1% NP-40, 0.1% SDS and 1% protease inhibitor cocktails). Cell lysates were incubated with biotin-labelled RNAs and streptavidin beads at 4ºC for 12 h. The beads were washed in wash buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 0.05% Nonidet P-40 (NP-40); 1 mM MgCl2) at 4°C six times. The samples were subjected to western blot analyses as described previously 31. Briefly, protein samples were denatured and subjected to SDS-polyacrylamide gel electrophoresis (SDS/PAGE), and were transferred to nitrocellulose membranes (Bio-Rad). The membranes were immunoblotted with specific primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and visualized by SuperSignal West Pico Stable Peroxide Solution (Thermo Fisher).

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