Figure 2.

In vitro interaction studies of Bat proteins with predicted DNA targets. (A) Electrophoretic mobility shift assays were carried out for Bat1, 2 and 3 using 5’Cy5 labelled double-stranded DNA, bearing target sequences deduced from the TALE code. Each protein (100 nM) was tested against each target DNA (10 nM). Cy5 fluorescence was visualized after running through a native polyacrylamide gel. A shifted band, running slower on the gel, indicates the protein–DNA complex. (B) The interaction between Bat1 and its target (BE_Bat1) was quantified using microscale thermophoresis. The fluorescence ratio over the thermophoretic jump is shown on the y-axis against DNA concentration. Standard deviation for four repetitions is indicated. Measurements were made with 40% LED and 20% laser power. The dark grey line indicates the Kd fit. (C) This was repeated for BE _Bat1 derivatives bearing A (grey bar), C (filled stripes) or G (spotted) at the zero position. The Kd was calculated in each case and is shown compared to that with BE_Bat1 (T₀, empty bar).