This data was developed using ab211498, the same antibody clone in a different buffer formulation.The IP was performed in a U-bottom non-adsorbing propylene 96-well plate. ab211498 (0.2 μg) was coated into Dynabeads^® sheep-anti-rabbit IgG (50 μl) for 1h at RT. Unmodified/modified oligonucleotides (5 μM) were added to samples containing the antibody/bead complexes and incubated with agitation for 1 hour at RT. After washing, Peroxidase-conjugated Streptavidin was incubated at 1/1000 dilution with agitation for 1 hour at RT. ECL substrate was then added and the results read in a non-transparent 96-well plate with a digital detector and analyzed using ImageJ. Lane 1: Buffer only. Lane 2: Modified oligonucleotide (5 μM), 5’ Biotin-mN.mN.mN.mN.mN.[m8A]*.mN.mN.mN.mN.mN 3’ Lane 3: Unmodified oligonucleotide (5 μM), 5’ Biotin-mN.mN.mN.mN.mN.[A]*.mN.mN.mN.mN.mN 3’ N - equimolar mixture of (A/U/G/C)
m - 2’O methyl protection
* - phosphorothioate protection Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: TBST/0.1% Triton X-100/1 mM EDTA

This data was developed using ab211498, the same antibody clone in a different buffer formulation.Biotinylated m8A (modified) and A (unmodified) oligonucleotides with the below sequence were coated onto wells of a 96 well plate. Modified oligonucleotide (5 μM), 5’ Biotin-mN.mN.mN.mN.mN.[m8A]*.mN.mN.mN.mN.mN 3’ Unmodified oligonucleotide (5 μM), 5’ Biotin-mN.mN.mN.mN.mN.[A]*.mN.mN.mN.mN.mN 3’ N - equimolar mixture of (A/U/G/C) m - 2’O methyl protection * - phosphorothioate protection ELISA was performed on 1.0 µg/ml of antigen using ab211498 at a concentration range of 0.005-4.000 µg/ml, followed by Goat Anti-Rabbit IgG, (H+L), alkaline phosphatase conjugated secondary antibody at 1/2500 dilution.