Specificity of N6-methyladenosine (m6A) (D9D9W) Rabbit mAb was determined by ELISA. The antibody was titrated against an RNA oligo containing either unmodified adenosine or N6-methylated adenosine (m6A). As shown in the graph, the antibody only binds to N6-methylated adenosine (m6A).
Specificity of N6-methyladenosine (m6A) (D9D9W) Rabbit mAb was determined by competitive ELISA. The graph depicts the binding of the antibody to a pre-coated m6A oligonucleotide in the presence of increasing concentrations of differentially modified adenosine. As shown in the graph, antibody binding is only blocked by free m6A nucleoside.
This antibody has been shown by an independent laboratory to work in RNA-IP-seq. Please use at an assay-dependent dilution.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
Note: This protocol is written for spotting either purified
total RNA or poly A-purified mRNA (titration of 2 μg, 1 μg, 500 ng,
250 ng, 125 ng, 62.5 ng, and 31.25 ng) onto a positively charged nylon
membrane using a 96-well dot blotting apparatus. Depending on the source of
the RNA, more or less RNA may be required for detection with the antibody.
• RNA is sensitive to degradation by RNases, which can affect sample integrity. It is recommended that all surfaces and equipment undergo RNase decontamination.
• Purify total RNA and/or mRNA from cell pellet using an RNA isolation kit. Assess total RNA quality by gel electrophoresis on a 1% agarose gel. The 28S and 18S RNA should present as distinct bands. Smearing indicates RNA degradation. See Figure 1.
• Cut a piece of nylon membrane to fit the size of the dot blot manifold.
• Wet nylon membrane with 10X SSC Buffer.
• Dry membrane by placing it in a 96-well dot blot apparatus and applying vacuum.
Optional: To normalize sample loading using methylene blue,
apply stain before Section C, Step 1 and capture an image. Rinse blots three
times for 5 min each with 15 mL dH2O. Stain does not affect
antibody binding or detection.
NOTE: Due to the kinetics of the detection reaction, signal is most intense immediately following incubation and declines over the following 2 hr.
Figure 1. Representative image of isolated, intact total RNA. 28S and 18S RNA should migrate as distinct bands. If RNA presents as a smear, the sample may be degraded and unfit to use in downstream assays. Lane 1 is NEB 100 bp DNA ladder and lane 2 is total RNA isolated from 293T cells.
posted November 2018
Protocol Id: 1784
N6-Methyladenosine (m6A) (D9D9W) Rabbit mAb recognizes endogenous levels of N6-methyladenosine (m6A). This antibody has been validated using ELISA and dot blot assays and shows high specificity for m6A. This antibody does not cross-react with unmodified adenosine, N6-dimethyladenosine, N1-methyladenosine, or 2'-O-methyladenosine.
All Species Expected
Monoclonal antibody is produced by immunizing animals with N6-methyladenosine.
N6-methyladenosine (m6A) is a post-transcriptional modification found in various RNA subtypes. While the presence of m6A in RNA was described decades ago, the lack of tools has made interrogating the epitranscriptomic landscape challenging (1,2). With the emergence of new technologies such as miCLIP and NG-RNA-seq, researchers have been able to show that m6A is a biologically relevant mark in mRNA that is enriched in 3’ UTRs and stop codons (3,4). The m6A writer complex consists of a core heterodimer of methyltransferase-like protein 3 (METTL3) and methytransferase-like protein 14 (METTL14), and the additional regulatory proteins Virlizer/VIRMA and Wilms tumor 1-associated protein (WTAP) (5). METTL3 is the catalytic methyltransferase subunit and METTL14 is the target recognition subunit that binds to RNA (6). The Virilzer/VIRMA protein directs m6A methylation to the 3’ UTRs and stop codons, and WTAP targets the complex to nuclear speckles, which are sites of RNA processing (7). Less is known about readers and erasers of m6A, and while the fat mass and obesity-associated protein FTO was the first discovered m6A demethylase, subsequent studies demonstrated that this enzyme may prefer the closely related m6Am mark in vivo (8,9). ALKBH5 was later shown to be a bona fide m6A demethylase enzyme, contributing to the idea that the m6A modification is dynamically regulated (10). Readers of the m6A mark include the YTH protein family, which can bind to m6A and influence mRNA stability and translation efficiency (3,11-13). The m6A mark and machinery have been shown to regulate a variety of cellular functions, including RNA splicing, translational control, pluripotency and cell fate determination, neuronal function, and disease (1, 14-17). The m6A writer complex has been linked to various cancer types including AML and endometrial cancers (18,19). Additionally, m6A has been implicated in resistance to chemotherapy (20).
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