Indirect ELISA and competitive ELISA results show that this antibody is specific to ac4C (N4-acetylcytidine). Indirect ELISA (blue curve, refer to top X-right Y axis) was performed by coating BSA conjugated ac4C at 50ng/well followed by blocking with 5% non fat milk. Serial diluted primary antibody was added to the plates and incubated at 37℃. HRP-goat anti-mouse was used for detection. Competitive ELISA was performed similarly except that different concentration of Ac4C or its structure analogue compounds was mixed in 20ng/mL primary antibody (red curve, refer to bottom X-left Y axis).

Total RNA was isolated from HeLa cell line and was dotted to NC membrane at different amount as indicated above the dots. The membrane was blocked with 5% milk and blotted with N4-acetylcytidine antibody 68498-1-Ig at 1:2000 followed by incubation of HRP-goat anti-mouse secondary antibody. Signal was developed by ECL substrate. A parallel dot blot was performed using Mouse IgG1 isotype control Monoclonal antibody 66360-1-Ig at the same dose.