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Fig. 1 | BMC Cancer

Fig. 1

From: An immunochemistry-based screen for chemical inhibitors of DNA-protein interactions and its application to human CGGBP1

Fig. 1

A schematic representation of the Dot-Blot and ImmunoDetection (DBID) assay. a: Genomic DNA was isolated from HEK293T cells. Sonicated DNA fragments (mean length of 1 kb) were blotted onto positively charged nylon membranes and crosslinked by vacuum heating at 80 °C. b: HEK293T cells were lysed and the cleared lysates were used as a source of protein for in vitro DNA binding. The assay depicted here is designed to detect binding of CGGBP1 (black circle) to DNA, although this protocol can be generically applied for any protein of interest. CGGBP1 can bind to DNA either directly (top panel) or indirectly (through linker proteins, depicted in the blue circle in the bottom panel). The subsequent immunochemistry-based detection reports a brown signal for the direct as well as indirect CGGBP1-DNA complexes alike. The immunochemistry employs a primary antibody against the protein of interest, a biotinylated secondary antibody, streptavidin-HRP conjugates and DAB as the chromogenic substrate and is semi-quantitative in nature. c: Pre-incubation of the cell lysate with inhibitors allows the small molecule compounds to bind to their cognate target proteins in the lysate. Only some of these compounds potentially inhibit the DNA-binding of their target proteins (exemplified with a yellow cross). d: The different possible outcomes of the DBID assay for inhibition of CGGBP1-DNA interactions are depicted. The direct inhibition of CGGBP1 (black circle with a cross) as well as the inhibition of a linker protein (blue circle with a cross) required for CGGBP1-DNA binding are expected to result in “No signal”. Inhibitors that do not have any direct or indirect effect on CGGBP1-DNA interaction show “Signal”

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