Abstract
Antibodies to N 6-methyladenosine were produced in rabbit by means of immunization with N 6-methyladenosine coupled to bovine serum albumin via periodate oxidation. Cross-reacting antibodies were removed by bovine serum albumin-Sepharose and appropriate nucleoside-human serum albumin absorbents. Nucleoside-specific antibodies were isolated by affinity chromatography on N 6-methyladenosine-human serum albumin-Sepharose. The specificity of the purified antibodies has been demonstrated by complement fixation inhibition analyses using nucleoside analogues as inhibitors. The homologous hapten was about 10 3 times more effective in inhibition than dAMP. Anti- N 6-methyladenosine was used to detect N 6-methyladenine in denatured DNAs from various sources by complement fixation. Practically no complement fixation has been found with DNAs containing no N 6-methyladenine, such as calf thymus, salmon sperm, Micrococcus radiodurans, Streptomyces chrysomallus and Streptomyces hygroscopicus, whereas a weak reactivity occurred in the case of Bacillus subtilis DNA and Sarcina maxima DNA. For DNA from Proteus mirabilis, Escherichia coli K-12, E. coli B, E. coli WF +, λ, T2 phages quantitative differences in the immunochemical reactivity were observed, which only partially correlate with the N 6-methyladenine content of the DNAs. Other factors, influencing the accessibility of N 6-methyladenine to the antibody-combining site have to be taken into consideration.
Published Version
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