Multiple Myeloma (MM) is characterised by malignancy of antibody-secreting B-cells that accumulate in the bone marrow . Tumour growth promotes hypoxia in the tumour environment. Hypoxic inducible factors (HIFS) are heterodimeric proteins composed of an inducible alpha-subunit and a constitutively expressed beta-subunit. In this experiment, the hypothesis being tested was if HIF1α and HIF2α regulate different genes in multiple myeloma. This was tested using the cross chromatin immunoprecipitation technique, coupled with the use of genomic sequencing. This technique offers to discern the sites at which the HIF1 and HIF2 proteins are binding to the DNA, and allow to infer the likelihood that these two proteins are regulating different genes in
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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