RNA pull down experiment for vA-202 (ANAPC1P2) lncRNA

ABSTRACT: This study aimed to identify protein interaction partners of the lncRNA vA-202 using an RNA pull-down approach followed by mass spectrometry. Biotinylated vA-202 RNA was generated by in vitro transcription incorporating biotin-14-CTP. For each pull-down reaction, 400 µg of whole-cell lysate (in a total volume of 100 µL) was incubated with 1 µg of biotinylated RNA in the presence of protease inhibitor cocktail (PIC), RNaseOUT, and 2× TENT buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 500 mM NaCl, 0.5% Tween-20), supplemented with IP lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol). The mixture was rotated at 4 °C for 2 hours. In parallel, streptavidin magnetic beads were washed three times with PBST (PBS + 0.1% Tween-20) and maintained on ice. The RNA-protein complexes were captured by adding the reaction mixture to the pre-washed magnetic beads and incubated for an additional 2 hours at 4 °C. Beads were then washed five times with PBST and twice with 100 mM ammonium bicarbonate. Bound proteins were eluted by incubating the beads in 0.1% RapiGest supplemented with 5 mM DTT at 60 °C for 30 minutes. The eluates were collected for downstream proteomic processing and LC-MS/MS analysis. Two controls were included: (i) a pull-down using an unrelated RNA (p-XEF) and (ii) a beads-only control (no RNA). All pull-down experiments were performed in triplicate.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

DISEASE(S): Malignant Neoplasm Of Ovary

SUBMITTER: Amruta Jadhav  

LAB HEAD: Dr. Sharmila A. Bapat

PROVIDER: PXD070150 | Pride | 2026-05-25

REPOSITORIES: Pride