Figure 2

Comparison of the classical EMSA and the DPI-ELISA. The specific binding to DNA is investigated with plant transcription factors of two classes: At bZIP63 (A.) and At WRKY11^DBD (B.). Specific binding of the recombinant proteins to double stranded (ds) DNA-probes by electrophoretic mobility shift assays (EMSA; left panel) or DPI-ELISA (right panel) is displayed. The sequences (top panel) of the dsDNA-probes are given in 5'-3'-orientation for the sense strand; changes of bases within the known binding consensi (bold face) are highlighted (red) in the mutated oligonucleotide versions [13, 14]. For EMSA, specific retardation bands are highlighted by red boxes; for the DPI-ELISA a picture of the respective plate-wells is displayed below each column of the histogram graph. A. Renatured At bZIP63 is tested with double stranded C- and Cm-probes. B. At WRKY11^DBD contained in crude protein extract from E. coli is tested with double stranded W2- and W2m-probes. Both experiments (A. + B.) confirm results from previous publications [13, 14]. C. Competition experiment: The specific binding of At WRKY11^DBD to W2-probes is competed with non-biotinylated dsDNA. Different amounts of W2- or W2m-probe (0, 2, 10, 50 pmol) were added to At WRKY11^DBD crude extract immediately prior the plate incubation. ELISA-plates are coated with 2 pmol of double stranded biotinylated W2^Bio-probe. The biotinylated dsDNA W2m^Bio-probe incubated with At WRKY11^DBD extract or the W2^Bio-probe incubated with BL21/RIL cells (transformed with an empty vector construct) serve as negative control.