10.7554/eLife.29118.015Figure 6—figure supplement 1.Free biotin prevents streptavidin binding to the biotinylated oligos but does not displace pre-bound streptavidin.To test the effectiveness of the free biotin trap used in Figure 6, the radiolabeled, dual-biotinylated oligos (0.5 nM) were incubated with streptavidin (4 μg/ml) and biotin (1.5 μM) was added before (lanes 3 and 10) or after (lanes 4–7 and 11–14) streptavidin incubation. Lanes 1/8 and lanes 2/9 are controls showing the migration of the substrates in the absence and presence of streptavidin, respectively, with no biotin addition. Lanes 3 and 10 demonstrate the effectiveness of the free biotin trap in preventing streptavidin binding to the substrates. Lanes 4–7 and 11–14 show that when the substrates are pre-incubated with streptavidin for 5’ at 30° C, addition of the biotin trap does not disrupt the pre-bound streptavidin over the 10’ time course used in the helicase assays in Figure 6. Below the gels is quantitation of the upper and lower bands in each lane of the gel by densitometry as described previously.