Primary antibody dilution: 1/500

Secondary antibody: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated 

Secondary antibody dilution: 1/20,000

Blocking buffer and dilution buffer: AdvanBlockTM Chemi Blocking buffer

Input: HeLa total RNA 0.5 µg per Dot

Competitive nucleosides: m1A, m2A, m2.2G

Exposure time: 37 seconds

m1A was immunoprecipitated from 20 μg of HeLa (Human cervix adenocarcinoma epithelial cell) polyA+ RNA with 10 μg of ab208196 and 40 μL of Protein G dynabeads per sample (the IP buffer was 50mM Tris-HCl pH 7.4, 150mM NaCl, and 0.1% NP-40). The amount of m1A was quantified relative to the level of G by LC-MS/MS with electrospray ionization and in positive ionization mode, and compared to the level of m6A/G in the same samples. Error bars represent technical replicate injections of the same sample in mass spec.

BSA-conjugated m1A (modified) and A (unmodified) nucleosides were coated onto wells of a 96 well plate. ELISA was performed on 1.0 µg/ml of antigen using ab208196 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.

Dot blot of total RNA using ab208196 at 2 ug/mL. The Amersham Hybond N+ membrane was pre-spotted with 500, 250, 125, 63 and 32 ng/dot of HeLa total RNA and 500ng of an unmodified RNA probe. The membrane was then blocked with 5% BSA in TBS with 0.1% Tween-20. Followed by blotting with anti-m1A ab208196, or ab208196 together with 100uM of free m1A nucleoside in the same blocking solution, to inhibit m1A binding. A goat anti-rabbit HRP was used as the secondary antibody at 1:5000 dilution. Methylene blue stain was used to verify RNA loading.

The IP was performed in a U-bottom non-adsorbing propylene 96-well plate.

ab208196 (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.[m1A].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