Chromatin Immunoprecipitation to Identify Target Protein Binding Sites on Genomic DNA
내레이션 대본
To identify the binding sites of a target protein on DNA, begin with oligodendrocyte precursor cells containing a target protein cross-linked to specific DNA sequences in the chromatin.
Treat the cells with a lysis buffer containing protease inhibitors. The lysis buffer lyses the cells, while the inhibitors inactivate proteases to retain the integrity of the protein-chromatin complexes.
Sonicate the content to shear the complexes into smaller chromatin fragments, with some retaining the target protein.
Centrifuge to pellet the cell components, and transfer the supernatant containing chromatin fragments into a fresh tube. Incubate with anti-target antibodies that specifically bind to the target protein on the chromatin fragments.
Supplement the tube with protein A-coated magnetic beads that bind to the Fc region of pre-bound antibodies. Apply a magnetic field to selectively precipitate the antibody-bound target proteins complexed with chromatin fragments.
Treat the bead complexes with a high salt-containing elution buffer to disrupt the protein-chromatin interactions and release the naked DNA without associated proteins.
Use organic solvents to extract the DNA. Add an amplification buffer, and run a polymerase chain reaction to amplify the DNA fragments. Post-amplification, analyze the DNA sequence.
Compare the sequence of the amplified products to the genomic DNA sequence of the cell. Find the region with a sequence match between the fragment and genomic DNA — correlating with the protein binding sites.
