DNase I footprinting Assay to detect DNA-protein interaction (CAT#: STEM-MB-0168-WXH)

This method uses DNase I to digest the radioactively-labeled or fluorescently-labeled DNA fragment, and then uses gel electrophoresis to detect the resulting cleavage pattern. The cleavage pattern of the DNA in the absence of a DNA binding protein, typically referred to as free DNA, is compared to the cleavage pattern of DNA in the presence of a DNA binding protein. If the protein binds to the DNA fragment, the binding site is protected from enzymatic cleavage. This protection will result in a clear area on the gel which is referred to as a “footprint”. By varying the concentration of the DNA-binding protein, the binding affinity of the protein can be estimated according to the minimum concentration of protein at which a footprint is observed.

• Detection of DNA-protein interaction.
• Identification of the exact binding sites of DNA-binding proteins.

1. Bind Protein to DNA Probe.
2. Add the appropriate amount of DNase I to cleave DNA. The amount of DNase I is controlled so that only one phosphodiester bond cleavage occurs for each DNA. Set up protein-free controls.
3. Remove the protein from the DNA, the modified DNA was loaded in the polyacrylamide electrophoresis gel and conducted autoradiography. The footprints of the nucleotide sequence are identified compared with the control group.