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