Typhoon trio image analyzer

The cleavage reactions for MacUDG-like and MacUDG were performed at 37 °C for 10 min in a 20 μL reaction mixture containing 50 mM Tris-HCl, pH 8.0, 1 mM DTT, 1 mM EDTA, 0.1 μg/mL BSA, 5 nM DNA, and various concentrations of proteins as described in the figure legends. The backbone of the DNA with an AP site was then cleaved by adding 2 μL of 200 mM NaOH and heating at 95 °C for 5 min, followed by adding 2 μL of 200 mM HCl for neutralization. The substrates were denatured with 20 μL of the stop solution and incubated at 95 °C for 5 min, followed by rapid cooling on ice. The samples were separated by 8 M urea-10% PAGE in TBE buffer. The gel image was visualized with a Typhoon TRIO+ image analyzer (GE Healthcare).

The cleavage reactions for MacExoIII were performed at 37 °C for various time periods in a 20-μL reaction mixture containing 50 mM Bis-Tris-HCl, pH 7.0, 1 mM DTT, 1 mM MnCl₂, 0.01% Tween 20, 5 nM DNA, and various concentrations of MacExoIII as described in the figure legends. The cleavage reactions for MacEndoQ were performed at 37 °C for 1 h in a 20-μL reaction mixture containing 50 mM Tris-HCl, pH 8.0, 1 mM DTT, 1 mM MgCl₂, 0.01% Tween 20, 5 nM DNA substrates, and various concentrations of MacEndoQ as described in the figure legends. The reactions were terminated with 20 μL of stop solution (98% deionized formamide, 10 mM EDTA, and 0.1% OrangeG). After incubation at 95 °C for 5 min, the samples were immediately placed on ice. The samples were separated by 8 M urea-12/15% PAGE in TBE buffer (89 mM Tris-borate and 2.5 mM EDTA). The gel image was visualized with a Typhoon TRIO+ image analyzer (GE Healthcare). Resulting band intensities were quantified with ImageQuant TL software (GE healthcare).