Gel shift assay of TrmBL2 binding to ssDNA and dsDNA.
The lanes contained TrmBL2 at increasing concentration (solid black ramp) and DNA. (A) Gel of ssDNA incubated with TrmBL2 at different concentrations. Marked with arrows are the ssDNA/TrmBL2 complex (top) and the ssDNA alone (bottom). (B) Gel of dsDNA incubated with TrmBL2 at different concentrations. Marked with arrows are the dsDNA/TrmBL2 complex (top) and the ssDNA alone (bottom). Note that unhybridized ssDNA runs at lower molecular weight. The bare dsDNA sample shows 2 bands, one annealed dsDNA band and one ssDNA band which resulted from unhybridised residual 60mer. The unhybridised band made up approximately 5% of the total fluorescence. (C) Titration curve using measured intensities of the bands indicate a KD of 2.0 μM for ssDNA. Data were fitted using a logistic binding model. (D) Titration curve after analysis of band intensities for TrmBL2 binding to dsDNA indicate a KD of 1.0 μM. Data were fitted using a Hill equation.
