The sample is then placed back into the centrifuge to isolate cellular contents. Once isolated, the cellular contents are sheared using sonication, the application of sound waves to agitate a media, into fragments between 200-1000 base pairs. Several samples are taken from the lysate and placed back into a centrifuge. The supernatant is collected and transferred to new test tubes. The protein of interest is then selectively enriched through the use of immunoprecipitation by introducing ChIP grade antibodies containing epitope(s) specific to the protein to the samples. These antibodies are complexed with either agarose or magnetic beads. These beads act as a matrix that allow for the efficient separation of DNA-protein fragments bound to antibodies and to those that are not. To isolate the protein of interest, a magnetic field or centrifuge, depending on the matrix, is coupled to the antibodies used during the immunoprecipitation. Once isolated, several washes with washing buffer are completed to minimise contamination of unwanted DNA fragments. Once the antibody-DNA-protein complex has been successfully isolated, the crosslinking needs to be reversed so DNA fragments corresponding to protein binding sites can be
The sample is then placed back into the centrifuge to isolate cellular contents. Once isolated, the cellular contents are sheared using sonication, the application of sound waves to agitate a media, into fragments between 200-1000 base pairs. Several samples are taken from the lysate and placed back into a centrifuge. The supernatant is collected and transferred to new test tubes. The protein of interest is then selectively enriched through the use of immunoprecipitation by introducing ChIP grade antibodies containing epitope(s) specific to the protein to the samples. These antibodies are complexed with either agarose or magnetic beads. These beads act as a matrix that allow for the efficient separation of DNA-protein fragments bound to antibodies and to those that are not. To isolate the protein of interest, a magnetic field or centrifuge, depending on the matrix, is coupled to the antibodies used during the immunoprecipitation. Once isolated, several washes with washing buffer are completed to minimise contamination of unwanted DNA fragments. Once the antibody-DNA-protein complex has been successfully isolated, the crosslinking needs to be reversed so DNA fragments corresponding to protein binding sites can be