N6-methyladenosine (m6A) monoclonal antibody - Classic

Figure 1. RNA immunoprecipitation using the Diagenode monoclonal antibody directed against m6A
RNA Immunoprecipitation was performed on 40 µg HeLa total RNA spiked with 0.5 µg of an in vitro prepared transcript containing 6mA nucleotides, using 2 µg of the Diagenode monoclonal m6A antibody (cat. nr. C15200082). An equal amount of IgG was used as negative control. The immunoprecipitated RNA was subsequently analyzed by qRT-PCR with primers specific for the transcript and for an intergenic region, used as negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

Figure 2. RNA immunoprecipitation using the Diagenode monoclonal antibody directed against m6A
Immunoprecipitation was performed on two radiolabelled synthetic DNA oligo’s, a 250 nt oligo containing m6A nucleotides and a 350 nt unmethylated control, using the Diagenode monoclonal m6A antibody (cat. nr. C15200082). The immunoprecipitated fraction is shown in lane 3; lane 2 shows the non bound fraction, whereas the input is shown in lane 1.

Figure 3. Dot blot analysis using the Diagenode monoclonal antibody directed against m6A
To demonstrate the specificity of the Diagenode monoclonal antibody against m6A (Cat. No. C15200082), a Dot Blot analysis was performed using an m6A containing, a non-methylated control and a non-methylated polyA control synthetic RNA oligonucleotide.

Figure 4. Dot blot analysis using the Diagenode monoclonal antibody directed against m6A
Dot Blot analysis was performed using a synthetic DNA oligonucleotide containing different m6A modified bases and a negative control. 100 to 4 pmol of the respective oligo’s were spotted on the membrane. The antibody was diluted of 1:500 in TBS-T containing 10 % skimmed milk and 1% BSA. Figure 4 shows a high specificity of the antibody for the modified oligonucleotide.