Description(s):

The chromatin immunoprecipitation (ChIP) assay is a major tool in the study of genomic processes and gene regulation in vivo. It is widely known control gene expression involves multiple proteins and great number of their modifications. Conventional ChIP protocols used agarose beads, which requires centrifugation for separation, blocking and lysate preclearing and may exhibit nonspecific binding.  Signosis has launched the magnetic bead based Snapshot ChIP assay plus multiple step optimization to reduce handling time and increase the reproducibility and sensitivity. 

 

Principle

The magnetic beads ChIP assay typically comprises four steps:  (1) cross-linking proteins to DNA; (2) chromatin fragmentation; (3) protein precipitation; and (4) target identification and quantitation.  The initial cross-linking is to insure that protein-DNA complexes remain associated through the subsequent steps. Formaldehyde is the most commonly used reagent for the cross-linking of proteins that are directly bound to DNA, such as transcription factors and histones, but not for coactivators and corepressors indirectly associated with DNA due to short spacer arm.

  

Several improvements in Signosis' ChIP assay are highlighted as follows:  firstly, the dual cross-linking, formaldehyde (short space arm) and second cross-linking reagent (long space arm), is introduced to ensure the detection of both TFs and associated cofactors.  Secondly, DNA is broken down to mononucleosomes and 500bp in size by MNase digestion, which enhances greatly the sensitivity and reproducibility.  The cross-linked protein-DNA complexes are subsequently pulled down by a specific antibody and ChIP grade protein A/G magnetic beads. The last improvement is to release DNA from cross-linked proteins with one-step chelex-100 without tedious proteinase K digestion and further purification, significantly increasing the assay efficiency.