Home Abstract Introduction
Cross-linking Cell Lysis and DNA Fragmentation Chromatin immunoprecipitation and elution of DNA cross-link reversal and DNA Purification preparation of DNA for Sequencing Next Generation Sequencing process
KLF1 peaks Distance of promoter peaks Analysis of KLF1 binding to gene body of ZBTB7A

Website for translation of

Laura J. Norton, Alister P.W. Funnell (2017)
KLF1 directly activates expression of the novel fetal globin repressor ZBTB7A/LRF in erythroid cells
Blood Advances 1:685-692

(Translated by Wassal Alhammad)

Experiment: Chromatin immunoprecipitation and elution of DNA

Third step is to precipitate the cross-linked and fragmented chromatin by adding antibodies specific to KLF1. Adding Anti KLF1 antibody to the crosslinked chromatin will specifically precipitate fragments of DNA that are crosslinked to KLF1 only. Precipitation can be enhanced by centrifugation or using magnetic beads.

The precipitated chromatin now is supposedly only those fragments of DNA that are crosslinked with KLF1 and bound to Anti-KLF1 antibodies.

After precipitation, elution is necessary to interrupt the interaction between the antibodies and the chromatin (Antigen-Antibody interaction). This can be achieved by adding elution buffer that will lead to disruption of the pH and hence breaking the Ag-Ab interaction

The result will be two different products: an antibody by itself and an antigen, KLF1-bound to DNA.

Fig. 6: Construction of Chromatin immunoprecipitation and elution of DNA. KLF1 antibodies added to fragmented chromatin to get antigen-antibody interaction (ag-ab, green-blue). by precipitation, the free chromatin was discraded and ag-ab complexes was eluted to result in antigens and antibodies saparated.