For the m6A dot blot assay, total RNA was extracted from cell aliquots using TRIzol, and the RNA samples were quantified by Nanodrop and UV crosslinked to nylon membranes. The membranes were blocked with 5% nonfat dry milk (in 1 × TBS) for 1 to 2 h and incubated with a specific anti-m6A antibody (1:2000, Proteintech) and mouse-HRP secondary antibodies. Then, the membranes were developed with ECL Western Blotting Substrate (Bio-Rad), visualized using Immobilon Western Chemilum HRP Substrate (Merck Millipore), washed once with TBST buffer for 5 min, and stained with MB for 30 min, followed by two or three washes with water. Subsequently, the relative m6A levels were determined by densitometry quantification and normalized to MB staining.
Anti m6a antibody
Manufactured by Proteintech
Sourced in China
The Anti-m6A antibody is a highly specific and sensitive tool used to detect the presence of N6-methyladenosine (m6A) modifications in RNA molecules. The antibody recognizes and binds to the m6A modification, allowing researchers to identify and quantify the occurrence of this important epigenetic mark in various RNA species.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon western chemilum hrp substrate, by Merck Group (1 mentions)
Ecl western blotting substrate, by Bio-Rad (1 mentions)
N6 methyladenosine 5 monophosphate sodium salt, by Merck Group (1 mentions)
Anti rabbit igg, by Abcam (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nylon n membrane, by GE Healthcare (1 mentions)
Anti mouse antibody, by Proteintech (1 mentions)
4 protocols using anti m6a antibody
1
Quantifying m6A levels by dot blot
2
Profiling m6A-modified IL-13 via MeRIP Assay
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Methylated RNA immunoprecipitation (MeRIP) assay was performed to examine m6A-modified IL-13. The total RNA from PAMs or Marc-145 cells was isolated using TRIzol reagent (Invitrogen), and 5 μg of anti-m6A antibody (Proteintech) or anti-rabbit IgG (Abcam) were conjugated to protein A/G magnetic beads. The eluted RNA was allowed to react with 200 μl of 0.5 mg/ml N6-methyladenosine 5-monophosphate sodium salt (Sigma-Aldrich) for 1 h at 4 °C. The immunoprecipitated RNA was reverse-transcribed to cDNA, and real-time PCR was subsequently performed with specific primers of IL-13.
3
m6A RNA Immunoblotting Protocol
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Total RNAs were spotted onto N+ nylon membranes (GE Healthcare, MD, USA). After ultraviolet cross-linking, the membranes were blocked with 5% fat-free milk in TBST for 1 h and then incubated with an anti-m6A antibody (1:1000, Proteintech, Wuhan, China) overnight at 4 °C. After washing, the membranes were incubated with an anti-mouse antibody (1:5000, Proteintech, Wuhan, China) for 1 h at 25 °C. After further washing, the membranes were incubated with enhanced ECL detection reagent (Biology, Wuhan, China) and visualized using a detection system. After washing, the membranes were stained with 0.2% methylene blue as a control.
4
m6A RNA Immunoprecipitation Protocol
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Total RNA was isolated using TRIzol reagent. The RNA was diluted to the indicated concentration, and the film was incubated with anti-m6A antibody (proteintech, #68055-1-Ig) at 4 °C. The nylon film was washed three times with TBST (5 min each). The nylon films were then incubated with sheep anti-rabbit IgG-HRP for 1 h. The nylon film was washed an additional three times for 10 min each time. Finally, ECL was loaded onto the nylon film and incubated in the dark for 5 min. Photographs were captured by a developer. The nylon film was placed in 10 ml methylene blue staining buffer for 30 min and then washed for approximately 30–60 s with double distilled water until the background was roughly clean. Images after methylene blue staining were collected using white-light imaging as a load control.
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