Native Chromatin Immunoprecipitation of Neurosphere Cells

Take neurosphere cells in a buffer containing detergents, calcium, and protease inhibitors.

The detergent permeabilizes cells, while the inhibitor inactivates proteases to preserve the chromatin-bound target protein.

Add nuclease and incubate. The nuclease uses calcium to digest the chromatin.

Add an EDTA-containing buffer that chelates calcium, inactivating the nuclease.

Add lysis buffer to lyse the cells, releasing the chromatin.

Centrifuge and collect the supernatant.

Add antibodies specific to the target protein and incubate with rotation. Centrifuge and remove the supernatant.

Introduce magnetic beads targeting the antibody and incubate with rotation.

Add lysis buffer to separate non-specifically bound DNA.

Magnetically capture the beads and remove the supernatant.

Add a high-salt buffer to separate non-specifically bound proteins.

Magnetically capture the beads and remove the supernatant.

Add a stabilization buffer, magnetically capture the beads, and remove the contaminants.

Resuspend in a proteinase-K buffer to digest the proteins, releasing the DNA.

Once the neurospheres are harvested, centrifuge 1 times 10 to the 6 neurosphere cells per immunoprecipitation at 300 times g for 5 minute