5-Methylcytosine Dot Blot to Determine the Extent of DNA Methylation

Start this procedure by denaturing the isolated DNA in 0.1 molar sodium hydroxide for 10 minutes at 95 degrees Celsius. Neutralize the DNA with 1 molar ammonium acetate on ice, and then dilute two-fold with double-distilled water.

The most critical step in the dot blot is the spotting of the samples onto the membrane, do it slowly, and carefully. Try to minimize each spotted area to 3 to 4 millimeters in diameter.

Using a narrow-mouth pipette tip, carefully spot 2 microliters of the serial diluted genomic DNA onto a positively-charged nylon membrane at the center of the grid. Blot the membrane at 80 degrees Celsius for 30 minutes. Next, block the non-specific antibody binding sites by soaking the membrane in 5% BSA in TBST in a 10-centimeter Petri dish for 1 hour at room temperature with gentle shaking.

After 1 hour, wash the membrane three times in TBST for 5 minutes each time. Incubate the membrane with a mouse anti-5-methylcytosine monoclonal antibody in TBST at 4 degrees Celsius overnight. On the following day, wash the membrane three times in TBST for 5 minutes each time.

Then, incubate with a secondary antibody — horseradish peroxidase-conjugated sheep anti-mouse immunoglobulin G in TBST for one hour at room temperature. After washing the membrane in TBST as before, add the enzyme substrate to the membrane, and incubate for 5 to 10 minutes. Finally, visualize the secondary antibody signal using a chemiluminescence kit according to the manufacturer's instructions.