Isolation and Normalization of DNA Containing Crosslinked Proteins

To begin the isolation and normalization of DNA-containing cross-linked proteins, aspirate the media using a suctioning pipette after ubiquitylation and SUMOylation drug treatment. Rinse the cells with ice-cold PBS before adding 600 microliters of DNAzol reagent. Slowly agitate the plate on a vibrating platform for 10 minutes at four degrees Celsius, and add 0.3 milliliters of 100%cold ethanol directly to the plate.

Repeat the agitation until opaque nucleic acid aggregate becomes visible. Next, transfer the cell lysate to a 1.5-milliliter microcentrifuge tube and centrifuge for 15 minutes at four degrees Celsius at 20, 000 G.After aspirating the supernatant, wash the nucleic acid and cross-linked protein pellet in one milliliter of 75%ethanol, followed by two minutes of centrifugation at 20, 000 G and four degrees Celsius. Aspirate the supernatant before spinning down at the same speed and removing the remaining liquid using a P-20 pipette.

Air-dry the pellet for five minutes. Dissolve the dried nucleic acid pellet in 0.1 milliliters of double-distilled water by pipetting up and down a few times. Then, incubate the suspension at 37 degrees Celsius in a water bath for 30 minutes until the pellet swells at least three times.

Sonicate the samples for 10 seconds using an ultrasonic processor probe at 30%amplitude and quantify the DNA concentration using a UV-Vis spectrometer. Add double-distilled water to adjust the concentration of the DNA to 400 to 500 nanograms per microliter in 120 microliters. Then, transfer 20 microliters of the sample to a new microcentrifuge tube as undigested DNA loading control.

Add 2, 000 gel units of micrococcal nuclease and 11 microliters of 10X calcium micrococcal nuclease reaction buffer to the DNA, dissolved in the remaining 100 microliters of double-distilled water. Incubate at 37 degrees Celsius for 30 minutes.