Indirect ELISA and competitive ELISA results show that this antibody is specific to 5-Hydroxymethylcytidine (hm5C). Indirect ELISA was performed by coating BSA conjugated 5-Hydroxymethylcytidine (hm5C) at 10ng/well followed by blocking with 1% BSA. 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 5-Hydroxymethylcytidine (hm5C) or its structure analogue compounds are mixed in primary antibody (fixed at 10 ng/mL).

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 BSA and blotted with 5-Hydroxymethylcytidine (hm5C) antibody 68579-1-Ig at 1:5000 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 antibody 66360-1-Ig at the same dose.