DNase I Footprinting Assay

DNase I footprinting assay is a detection method that accurately identifies the binding sites of DNA binding proteins (such as transcription regulatory proteins) on DNA molecules. Transcription regulatory proteins regulate the transcription of target genes by binding to their promoter regions. At present, the EMSA (Electrophoretic Mobility Shift Assay) and DNase I footprinting assay are important sets of in vitro research tools. EMSA determines whether transcription factors are directly bound to DNA, while DNase I footprinting assay can accurately identify the DNA sequence to which they bind, thereby helping us to study the mechanism of gene transcription regulation.

The classic DNase I footprint analysis experiment requires the use of isotopes for labeling, which is not only dangerous, but also time-consuming, requires high technical requirements for the operator, and has a low success rate. In this case, our company uses "fluorescent labeling" combined with the ABI sequencer method to perform DNase I footprinting assay. The DNA can be accurately quantified, and the experiment can be operated on a conventional laboratory bench. The repeatability of the fluorescent labeling method is much better than isotope operation. Moreover, the operating system of ABI sequencer is very mature, with high sensitivity and good repeatability. Therefore, our DNase I footprinting assay is more sensitive, the protected area obtained is clearer, and the quality of the experimental picture is better.

Figure 1. The principle of DNase footprinting assay and comparison between isotope labeling method and fluorescent labeling method.

Lifeasible provides DNase footprinting testing service with high quality and short turnaround time. The experimental operation process of DNase footprinting assay is as shown below.

Figure 2. The experimental operation process and lead time of DNase I footprinting assay.

Information customer needs to provide:

• T vector with target DNA sequences. OD260/280 of DNA should be about 1.8 (1.7～2.0); DNA concentration is > 30 ng/µL; the total amount of DNA is not less than 1 µg.
• PCR amplification primers for target probes. The length of the target probe is 150-300 bp.
• Purified specific protein. The purity of protein sample is required to be no less than 90% (Generally the purity of protein is 90% when electrophoresis shows a band on PAGE gel); The concentration should not be less than 300 ng/μL; The total amount is generally no less than 30-300 g (depending on the binding ability of protein and DNA). Protein is recommended to be divided into multiple tubes and be stored at -80°C, and cannot be repeatedly frozen. Samples are required to be transported immediately on dry ice without thawing. The concentration of NaCl in protein buffer must be less 150 mM and the protein buffer must have no imidazole. If you can't provide required protein, our company can be responsible for protein expression and purification services.
• DNA-protein binding reaction conditions. Customer can specify the DNA-protein binding reaction conditions.

Delivered result: Detailed experimental procedures, Experimental raw data, DNase I footprinting assay picture, and complete experiment report.

Lifeasible has been deeply involved in the field of molecular biology and protein detection for many years, and has achieved gratifying scientific research results in DNase footprinting analysis. Our company's latest analysis technology can help customers obtain satisfactory experimental results and publishable paper diagram. Please don't hesitate to contact us.