This article details a protocol for chromatin immunoprecipitation (ChIP) using magnetic beads coated with antibodies specific to target proteins. The method involves several washing and centrifugation steps to isolate protein-DNA complexes from a motor neuron lysate.
Begin with a tube containing pre-treated magnetic beads coated with antibodies specific to a target protein cross-linked to the DNA.
Add a motor neuron lysate containing DNA fragments and incubate with agitation.
The DNA fragments interact with antibody-coated beads, forming bead-DNA complexes.
Centrifuge to collect the liquid from the cap and place it on a magnetic separator to precipitate the bead-DNA complexes.
Remove the supernatant containing unbound DNA fragments.
Introduce a detergent-based lysis buffer to solubilize non-specifically bound DNA fragments.
Centrifuge and perform the magnetic separation, then remove the supernatant.
Wash with a high salt buffer to disrupt non-specific interactions. Centrifuge, perform the magnetic separation, and remove the supernatant.
Wash with LiCl buffer to remove the remaining non-specific interactions. Centrifuge, perform the magnetic separation, and remove the supernatant.
Finally, wash with Tris-HCl buffer, centrifuge, and perform the magnetic separation.
Remove the supernatant and collect the beads containing the target protein-DNA complexes.
To begin the chromatin immunoprecipitation, wash the antibody-coated beads in 1 milliliter of blocking solution. Place the tube containing the antibody-coated beads on a rocking platform at 4 degrees Celsius for 5 minutes. After briefly spinning, place the tube on a magnetic rack, and remove the supernatant.
Resuspend the antibody-coated beads in 50 microliters of blocking solution. Add 1 milliliter of sonicated lysates to the antibody-coated beads. Then, incubate the sample on a rocking platform at 4 degrees Celsius overnight. After allowing the sample to incubate overnight, collect liquid from the cap by briefly spinning the tube. Then, place the tube on a magnetic rack, and after 1 minute, remove the supernatant carefully with a pipette.
Next, perform a series of washes as follows. Add 1 milliliter of cold wash buffer containing CPI to the sample. Mix the sample on a rocking platform at 4 degrees Celsius for 5 minutes. Briefly spin the sample tube. Then, place on a magnetic rack. After 1 minute, remove the supernatant with a pipette.
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