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. Author manuscript; available in PMC: 2016 Sep 17.
Published in final edited form as: Mol Cell. 2015 Sep 17;59(6):1025–1034. doi: 10.1016/j.molcel.2015.08.012

Figure 3. EMSA of DNA Binding by Core7.

Figure 3

(A) Core7 has different affinity for ssDNA (45 nt, ss45) and dsDNA (45 bp, ds45). The 5′-32P-labeled DNAs (0.5 nM) were incubated with Core7 (0, 0.25, 0.5, 1, 2, 4, 8, 16, and 20 nM). Two protein-DNA complexes were detected with ss45 (1 and 2).

(B) Different lengths of 32P-labeled ssDNAs (0.5 nM) were incubated with Core7 (0, 0.5, 4, and 20 nM). The longer the ssDNA used, the more the protein-DNA complexes formed.

(C) The same EMSA assays as shown in (A) were performed for ssDNA of variant lengths, and the results were quantified and plotted. Dissociation constants (KD) were calculated by fitting the EMSA data with the non-linear regression model (Prism) and shown in the table next to the plot. Error bars were estimated from triplicate measurements.

(D and E) Effects of Cy5 (D), CisPt (E), and ATP on DNA binding by Core7. Core7 (0.5, 1, 4, and 20 nM) and ssDNA were incubated with or without ATP and MgCl2 and analyzed by EMSA. The percentage of Core7-DNA complexes in each lane was quantified and indicated below the gels.