| Description | I. Experimental principle DNA pull down technique is a powerful tool for studying DNA-protein interaction in vitro. In this technique, specific DNA probes are designed for the target region and labeled with desulfuryl biotin. After labeling, the labeled probes can bind to the streptavidin conjugated to the magnetic beads, and then incubated with the total protein extract. The non-specific binding proteins were removed by washing, and then the target DNA probe-protein complexes were obtained by elution. Finally, the protein types were identified by Western Blot or mass spectrometry (MS). The schematic diagram is as follows:
2, experiment flow chart
3, the flow chart of control group Settings
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| Composition | Component | Size | Storage | Nucleic-Acid Compatible Streptavidin Magnetic Beads | 270uL | 4°C | RNA binding buffer | 6mL | 4°C | Wash Buffer I | 20mL | 4°C | Protein-RNA binding buffer (10×) | 600uL | 4°C | IP lysis buffer | 9mL | 4°C | Protease inhibitor(100×) | 35uL | -20°C | Glycerol (50%) | 800uL | 4°C | Wash Buffer II | 10mL | 4°C | Solution I | 600uL | 4°C | Elution Buffer | 800uL | Avoid light at 4°C | DEPC H2O | 400uL | 4°C |
Note:
1, need to bring your own Trizol, 75% ethanol, chloroform, isopropyl alcohol, reverse transcription kit and fluorescent dye;
2 and 6T were six single-group (one experimental group or one control group) immunoprecipitation experiments. In the later operation steps, one experimental group and one control group were included, and 2T reagent was needed. |
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