Principle

SMAD transcription factors are the core members of transforming growth factor β (TGF-β) pathway, which has been implicated in the regulation of cell growth, differentiation, apoptosis and specification of developmental fate. The molecular mechanisms of TGF-β/SMAD signaling pathway have been widely studied. The regulatory effect of SMAD complex likely depends on transcriptional modules, in which the SMAD binding elements and partner transcription factor binding sites (SMAD modules) are present in specific context on the promoter regions.

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 Smad2/3  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' Smad2/3 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.