Figure 5.

Quantification of aldehydic lesions and AP sites using ARP and AA3. (A) HeLa DNA was labeled with ARP and vacuum-spotted onto a nylon membrane. Aldehydic lesions in this DNA were quantified using streptavidin-conjugated horseradish peroxidase (HRP) and detected using a chemiluminescent substrate (open bar). In parallel, aldehydic lesions in HeLa DNA were also labeled with AA3 followed by reaction with Cy5 azide and quantification of fluorescence on a nylon membrane (black bar). AA3-labeled aldehydic lesions reacted with Cy5 azide were also quantified in solution using a microplate reader (gray bars). Mean and standard deviation of triplicate samples is shown. (B) HeLa DNA was pre-treated with NaBH₄ to reduce endogenous aldehydic lesions and AP sites were generated by heat and acid treatment for different lengths of time. The AP sites were labeled using ARP or AA3 for labeling and the DNA was spotted onto a nylon membrane and the membranes scanned to quantify AP sites (Left). AP sites in the same DNAs were also quantified by reacting them successively with AA3 and Cy5 azide, and measuring fluorescence intensity directly using a microplate reader (right). Mean and standard deviation of triplicate samples is shown for each time point.