Figure 3

Validation of microplate-based MeDIP assay. A , Denatured HeLa cell DNA was preincubated with either monoclonal anti-5mC or Flag (mock control) monoclonal antibody (0.5 μg each) in binding buffer (60 μl, 60 minutes at 4°C) in ultrasonic bath. After antibody preincubation, the mixture was divided into two 30 μl aliquots, one was used in microplate MeDIP (Microplate MeDIP) and the other in protein A-agarose beads MeDIP (Beads MeDIP). For each step the same buffer volumes were used in microplate- and beads-based assays. The X-axis shows total DNA input used per IP (nanograms DNA/30 μl IP reaction). After binding, wells and beads were washed with IP buffer and TE buffer. DNA was purified from well walls using 60 μl elution buffer/proteinase K and from beads using 60 μl 10% Chelex/proteinase K [46]. Eluted DNA was used in real-time PCR using primers to ALU and LINE elements. Results are expressed as % Input, mean ± SEM of three independent biological replicates. B , As in (A) microplate and beads immunoprecipitated DNA was used in real-time PCR using primers to either the methylated H19 imprinted control region (H19 ICR) or the unmethylated promoter region of the house keeping gene UBE2B [18]. Data are expressed as ratio of the 5mC signal to the Flag mock (5mC/Flag). The red horizontal line represents background binding, 5mC/Flag ratio = 1.