Protocol to detect nucleotide-protein interaction in vitro using a non-radioactive competitive electrophoretic mobility shift assay

Figures

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  Figure 1. A figure illustrated the schematic diagram of the EMSA method Target protein and DIG-labeled DNA probe are co-incubated under appropriate conditions for protein-DNA reaction (left panel). The reaction mixtures without (1) or with (2) target proteins are loaded into a native PAGE gel, and immunoblotted to detect protein-DNA interactions (middle and right panel). A retarded gel shifting band is expected if the target protein has binding activity to the tested DNA probe.

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  Figure 2. A representative figure displaying the wet-transfer procedure

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  Figure 3. A figure illustrated the schematic diagram of the competitive EMSA assay Target protein, DIG-labeled DNA probe and 50–100 fold more unlabeled DNA probe are co-incubated at specific conditions for reaction (left panel). The reaction mixtures without (1) or with (2) unlabeled DNA probe are loaded into a native PAGE gel, and immunoblotted to detect competitive protein-DNA interaction (middle and right panel). Those unlabeled DNA probe effectively compete for the binding of the target protein to the DIG-labeled DNA probe, and the retarded band is expected to disappear if the target protein has specific binding activity to the tested DNA probe.

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  Figure 4. DNA labeling efficiency detection The DIG labeling efficiency for the promoter region of ler (top panel) and rpoS fragment (bottom panel) were detected and indicated, respectively. The immunoblot displayed here was captured after 20 s of exposure. The content of each probe was shown at the bottom of each lane.

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  Figure 5. The EMSA detection for the target protein AirA with ler promoter and the negative control rpoS The reactions of different concentrations of protein AirA with the promoter region of gene ler (left panel) and control gene rpoS (right panel) were loaded, and figures were displayed after immunoblotting, respectively. In each lane, the DNA probe level is confined (i.e., 1 nM), together with a gradually increased AirA concentration, accordingly. B and F represent the bound and free DNA fragments, respectively. The concentrations of AirA and DIG-labeled probes were indicated at the bottom of each blot. The blots displayed here were captured after 15 s of exposure.

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  Figure 6. The binding of target protein AirA to ler promoter is not affected by non-specific DNA competitor poly [d(I-C)] Poly [d(I-C)] was added in the same protein-DNA reactions as described in Figure 5. A native PAGE gel running and immunoblotting were further performed and the immunoblot figures were displayed. In each lane, the DNA probe level (ler promoter or rpoS) (i.e., 1 nM) and poly [d(I-C)] (i.e., 50 ng/μL) are confined, together with a gradually increased AirA concentration, accordingly. B and F represent the bound and free DNA fragments, respectively. The concentrations of protein AirA, DIG-labeled probes and poly [d(I-C)] were indicated at the bottom of each blot. The blots displayed here were captured after 15 s of exposure.

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  Figure 7. The competition EMSA result for the protein AirA with ler promoter and the negative control rpoS Selected concentrations of protein AirA (i.e., 0, 30 and 60 nM) with 1 nM DIG-labeled DNA probe and a range of unlabeled DNA concentrations (i.e., 0, 10, 50, 100× times more) in each lane were loaded accordingly, for both target gene ler (left panel) and the control gene rpoS (right panel). B represents the AirA bound DNA fragments and F represents the free DNA fragments. The concentrations of protein AirA, DIG-labeled probes and unlabeled probes were indicated at the bottom of each blot, respectively. The blots displayed here were captured after 15 s of exposure.