Argonaute-2 Immunoprecipitation for RNA Binding

RNA binding protein immunoprecipitation (RIP) assay was executed by using Magna RNA RIP Kit (Merck KGaA, Darmstadt, Germany) by following the manufacturer’s protocol. INS-1E cells were lysed by using RIP lysis buffer (Beyotime) and incubated with magnetic beads conjugated with Argonaute-2 (Ago2, Millipore, MA, USA) or control immunoglobulin G (IgG, Millipore) at 4ʹC overnight. Detachment of the complex was with proteinase K (Sigma-Aldrich), and the bound RNA was identified by RT-qPCR [14 (link)].

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, & Ren L. (2022). Circular RNA PIP5K1A act as microRNA-552-3p sponge to regulates inflammation, oxidative damage in glucolipotoxicity-induced pancreatic INS-1 β-cells via Janus kinase 1. Bioengineered, 13(3), 5724-5736.

Publication 2022

Magna RNA RIP Kit RIP lysis buffer Argonaute-2 control immunoglobulin G (IgG, proteinase K

Ago2 Assay Buffer Cells Immunoglobulin g Immunoprecipitation Proteinase k Rna binding protein

Corresponding Organization : First Affiliated Hospital of Zhengzhou University

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Variable analysis

independent variables

Argonaute-2 (Ago2) magnetic beads
Control immunoglobulin G (IgG) magnetic beads

dependent variables

Bound RNA identified by RT-qPCR

control variables

INS-1E cell line
RIP lysis buffer (Beyotime)
Proteinase K (Sigma-Aldrich)
Incubation at 4°C overnight

controls

Positive control: Argonaute-2 (Ago2) magnetic beads
Negative control: Control immunoglobulin G (IgG) magnetic beads

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