Proteins synthesized in vitro are extremely useful for determining whether a cloned gene encodes a specific DNA-binding protein and for analyzing DNA-protein interactions. To test DNA binding capacity, the labeled protein can be co-conditioned with the specific DNA fragment and the protein-DNA complex can be separated from the free protein by non-denaturing acrylamide gel electrophoresis. Source: Comprehensive Molecular Biology Laboratory Guide, 5th edition.
Operation method
basic program
Materials and Instruments
Plasmid DNA containing the desired binding site Move 1) Cut 0.5 μg of plasmid DNA containing the binding site (assuming a 5 kb plasmid) with a suitable restriction endonuclease to produce a fragment 150 ~ 700 bp in length, which is different in size from the size of the other fragments cut by the endonuclease. Purify by phenol extraction and ethanol precipitation. 2) Create the following 15 μL binding reaction: 5 μL H2O 3 μL 5× binding buffer 5μL DNA (9 nmol/L for each DNA fragment) 1μL 10 mg/mL poly (dl-dC) . poly (dl-dC) 1 μL 35S-labeled protein Incubate for 20 min at room temperature. Set up control reactions containing no DNA and containing non-specific DNA (i.e., lacking binding sites). 3) Add 5 μL of spiking buffer and immediately spike onto a 5% non-denaturing polyacrylamide gel. Perform electrophoresis until the bromophenol blue is close to the bottom of the gel (about 1~4 h depending on the buffer conditions), and do not allow the temperature of the gel to rise above room temperature. The composition of the gel and the ionic strength of the buffer can be changed, and these changes may be important for detecting protein-DNA interactions. 4) After electrophoresis, the gel was fixed in 45% methanol/10% acetic acid for 1 h at room temperature. The gel was treated with EN3HANCE for 1 h, dried, and then subjected to radioautoradiography. Common Problems Unlike the more commonly used mobility shift assays that use 32P-labeled DNA and unlabeled proteins, the assays presented here generally use 35S-labeled proteins and unlabeled DNA.The main advantages of this method are that the DNA binding properties of any mutant protein can be determined by changing the DNA template, and that subunit structures (e.g., dimers. tetramers) can be detected, tetramers) For more product details, please visit Aladdin Scientific website.
35S-tagged protein 5× binding buffer 10 mg mL poly (dl-dC) - poly (dl-dC) or other macromolecular carrier DNA spiking buffer 45% methanol 10% acetic acid EN3HANCE (Du Pont NEN)