Isolation of Proteins from Viral DNA-protein Complexes

Take the cell nuclei containing viral genomes, labeled with alkyne-modified nucleotides.

Suspend the nuclei in a mixture of biotin-azide and copper ions, and incubate.

The copper ion aids in the covalent bonding of the azide and the alkyne group.

Centrifuge and discard the supernatant.

Resuspend in buffer, transfer, and centrifuge to pellet.

Flash-freeze, then thaw the pellet to aid in lysis.

Resuspend and sonicate to lyse the nuclei.

Centrifuge and collect the supernatant containing protein-viral DNA complexes.

Filter the supernatant to remove contaminants.

Dilute the filtrate.

Add streptavidin-coated magnetic beads and incubate to facilitate binding with biotinylated protein-DNA complexes.

Apply a magnetic field to collect the beads, and wash to remove contaminants.

Repeat the washing.

Add an elution buffer and heat to separate and denature the proteins.

Separate the beads, collect the eluate.

Flash-freeze the isolated proteins. Store at ultra-low temperatures for further analysis.

After completely removing the PBS, re-suspend the nuclear pellet in 10 milliliters of click reaction mix, by gently pipetting up and down five times with a 10 milliliter pipette. Rotate the sample for one hour at four degree celsius, before pelleting the nuclei as before.

Completely remove the click reaction mix, then, wash the pellet by gently re-suspending it in 10 milliliters of PBS and centrifuging again. Now, carefully re-suspend the nuclear pellet in one milliliter PBS and transfer the solution to a 1.5 milliliter microcentrifuge tube. Following centrifugation, completely remove the PBS and flash freeze the pellet in liquid nitrogen.

At this point, the pellet can be stored at 80 degree celsius or the procedure can be continued. To lyse the nuclei and fragment the DNA, re-suspend the thawed nuclei in 500 microliters of Buffer B1 by pipetting up and down. Incubate the suspension on ice for 45 minutes.

Then, sonicate the samples six times for 30 seconds each at 40%amplitude, using a three millimeter microtip probe. Place the samples on ice for at least 30 seconds between pulses. After sonication, samples should appear clear, not cloudy.

Pellet the cell debris by centrifuging at 14, 000 x G for 10 minutes at four degrees celsius. The pellet size should decrease substantially, filter the supernatant through a 100 micron cell strainer and retain the flow through. Then, add 500 microliters of Buffer B2 to the filtered supernatant.

To bind the biotinylated DNA to streptavidin coated beads, prepare the streptavidin magnetic beads by transferring 300 microliters of bead slurry to a 1.5 milliliter microcentrifuge tube. Prepare one tube of beads per sample. Wash the beads three times with milliliter of Buffer B2 by vortexing to re-suspend, applying to a magnet to separate the beads and aspirating the wash buffer.

Then, add 900 microliters of the sample to the washed beads. The remainder of the sample will be used for input DNA and protein isolation. Rotate the suspension overnight at four degree celsius.

The next day, place the samples into a magnetic microfuge tube rack and remove the supernatant. Gently re-suspend the beads in one milliliter of Buffer B2, before rotating at four degree celsius for five minutes. Remove the supernatant again using the magnetic microfuge tube rack.

This time, gently re-suspend the beads in one milliliter Buffer B3 and rotate at four degree celsius for five minutes. After removing the supernatant from the bead mixture using the magnet, re-suspend the beads in 50 microliters of 2x Laemmli sample buffer to elute DNA protein complexes. Next, boil the samples at 95 degrees celsius for 15 minutes.

Vortex a mix before quickly spinning the samples in a microfuge. Apply the samples to the magnet and transfer the eluate to a new tube. Finally, flash freeze the samples and store them at 80 degrees celsius.

Perform protein analysis as described in the text protocol.