RIP was performed using an EZ-Magna RIP Kit (Millipore) according to the manufacturer’s protocol. Briefly, the whole-cell lysate was incubated with RIP buffer containing magnetic beads conjugated to an anti-Ago2 antibody (Millipore) or anti-m6A antibody (Epigentek, Farmingdale, NY, USA) at 4 °C for 6 h. Immunoglobulin G (IgG) was used as the negative control. The beads were washed and were then incubated with 0.1% SDS/0.5 mg/mL proteinase K at 55 °C for 30 min to remove proteins. Finally, the coprecipitated RNAs were evaluated by qRT-PCR.
Anti m6a antibody
Manufactured by Epigentek
Sourced in United States
The Anti-m6A antibody is a laboratory reagent used for the detection and study of N6-methyladenosine (m6A) modifications in RNA. It specifically binds to the m6A modification, allowing researchers to investigate the distribution and regulation of this important epigenetic mark.
Automatically generated - may contain errors
3 protocols using anti m6a antibody
1
RNA Immunoprecipitation for m6A Detection
2
RNA-Binding Protein Immunoprecipitation Assay
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The RIP assay was executed using RNA-Binding Protein Immunoprecipitation Kit (17–700, Millipore) and HiScript II qRT SuperMix II kit (R223, VAZYME). Anti-m6A antibody (A-1801–020, Epigentek) and anti-Ago2 (ab5072, Cambridge, MA, USA) were used for RIP and MeRIP assays.
3
Quantifying m6A-enriched RNA in LOVO cells
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Total RNA was isolated from LOVO cells. According to the standard operating protocol for the EpiQuik™ CUT&RUN m6A RNA Enrichment Kit (Epigentek, NY, USA), RNA samples were fragmented and incubated with anti-m6A antibody (#A-1801, Epigentek) for 90 min at room temperature. A nonimmune IgG was used as a negative control. RT-qPCR assays with DSN1 primers were performed to quantify the enrichment of m6A-containing RNA. Information on the primer sequences used in this study is summarized in Additional file 2 : Table S2. Each experiment was performed in triplicate, and all samples were normalized to β-actin.
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