Anti cpd and anti 6 4pp antibodies

An ELISA was used to measure the amount of CPD and 6-4PP present in UV damaged DNA using anti-CPD and anti-6-4PP antibodies (Cosmo Bio Co. Ltd., Japan) following manufacturer's instructions. Briefly, 96 well polyvinylchloride plates (Thermo Cat. No. 2801) were coated with 50 μl of 0.003% protamine sulfate overnight at 37°C. Plates were washed with 100 μl distilled water and stored in the dark until used. Plates were coated with DNA obtained from ChIP or directly isolated from UV treated cells (0.02 μg/ml for CPD and 2 μg/ml for 6-4PP) for 30 min at 37°C. Plates were washed 5 times with PBS-T (0.05% Tween-20 in PBS), blocked with 2% FBS in PBS, incubated with appropriate dilutions of anti-CPD and anti-6-4PP antibodies, detected with biotin-F (ab)2 fragment of anti-mouse IgG (H+L) and peroxidase-streptavidin using o-phenylene diamine as a substrate.

Repair of UVB-induced damage was determined by measuring the amount of 6-4PP and CPD remaining in genomic DNA over time as previously described (19 (link)). Briefly, the dorsal skins of mice were shaved 24 h prior to a single UVB dose of 1,000 J/m². At various time points post-UVB, epidermis from the exposed skin was collected and genomic DNA was isolated using Sigma GenElute mammalian genomic DNA isolation Kit. Equal amounts of DNA were coated onto 96 well microtiter plate pre-coated with protamine sulfate. The DNA photoproducts CPD and 6-4PP were detected by ELISA using anti-CPD and anti-6-4PP antibodies (Cosmo Bio Co. Ltd., Japan) following manufacturer's protocol. Two mice for each genotype and time point were analyzed.