Anti-1-methyladenosine (m1A) antibody [EPR-19836-208]

• IP

Collaborator

Immunoprecipitation - Anti-1-methyladenosine (m1A) antibody [EPR-19836-208] (AB208196)

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.

All lanes:

Immunoprecipitation - Anti-1-methyladenosine (m1A) antibody [EPR-19836-208] (ab208196)

false

This image is courtesy of Dr Sigrid Nachtergaele, University of Chicago.

• ELISA

Supplier Data

ELISA - Anti-1-methyladenosine (m1A) antibody [EPR-19836-208] (AB208196)

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.

• IP

Supplier Data

Immunoprecipitation - Anti-1-methyladenosine (m1A) antibody [EPR-19836-208] (AB208196)

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

All lanes:

Immunoprecipitation - Anti-1-methyladenosine (m1A) antibody [EPR-19836-208] (ab208196)

true

• Dot

Collaborator

Dot Blot - Anti-1-methyladenosine (m1A) antibody [EPR-19836-208] (AB208196)

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.

This image is courtesy of Dr Sigrid Nachtergaele, University of Chicago.

• Dot

Lab

Dot Blot - Anti-1-methyladenosine (m1A) antibody [EPR-19836-208] (AB208196)

Primary antibody dilution : 1/500

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

Secondary antibody dilution : 1/20000

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