Principle

HIF-1 functions as a master regulator to balance oxygen supply and demand. HIF-1 is also activated in cancer cells by tumor suppressor (e.g., VHL) loss of function and oncogene gain of function (leading to PI3K/AKT/mTOR activity) and mediates metabolic alterations that drive cancer progression and resistance to therapy. HIF binds to the consensus sequence 5′-RCGTG-3′ that is present within or near the promoter regions of HIF-1–regulated genes.

Unlike electrophoretic mobility shift assays detecting the binding of specific TFs present in cell lysates to DNA sequences in vitro, chromatin immunoprecipitation (ChIP) assay is enable the analysis of the association of HIF with specific promoters in vivo and provides a snapshot of how a regulatory TF or co-factor affects the expression of a single gene or a variety of genes at the same time.  With several improvements, Signosis' HIF ChIP assay is able to efficiently measure the interaction of a specific TF or associated cofactors with target promoters in human and mouse samples.

The assay utilizes the surface-immobilized antibodies in two 8-well strips. The entire procedure from chromatin precipitation to PCR-ready DNA is done on the same wells without sample transfers. The microplate is first blocked and coated with protein A&G, The plate is then coated with one or more antibodies of interest. The cells in 96-well culture plate or any size of dishes are cross-linked. After chromatin fragmentation, the cell lysates are transferred and incubated on the antibody-coated plate to form antibody-protein-DNA complex. After several wash steps, the bound DNAs are subsequently eluted. The eluted DNAs can be used for PCR analysis or next generation sequencing. The microplate-based ChIP assay has several important advantages over the tube-based assay: simple sample handling, high throughput, improved sensitivity and reproducibility.