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. 2014 Oct 27;42(20):12912–12927. doi: 10.1093/nar/gku960

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© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 7. — Binding of Tim-Tipin-RPA to ssDNA substrates. (A) EMSA using 60 and 14 nt 5′-FAM labeled ssDNA substrate. The protein-ssDNA complexes were visualized by fluorescence imaging (EMSA), immunoblot analysis using an anti-RPA70 and anti-Tipin primary antibody and Coomassie stain. In the presence of long excess ssDNA significant amounts of intact Tim-Tipin-RPA-ssDNA complex were detected as indicated by the left square. The presence of substoichiometric amounts of long ssDNA (lanes 4 and 5) and short ssDNA (lanes 6–10) lead to dissociation of the Tim-Tipin-RPA-ssDNA complex into DNA-free Tim-Tipin and DNA-bound RPA. The total ssDNA concentration was varied from 64, 32, 8 to 4 μM as indicated by (). The Tim-Tipin-RPA concentration was kept constant at 16 μM. R, RPA; T, Tim-Tipin; P, pentameric Tim-Tipin-RPA. Lanes 11–13 show the DNA-free proteins. (B) EMSA after cross-linking the complex. Double excess amounts of 60 nt 5′-FAM labeled ssDNA and RPA (3.12 μM:1.56 μM RPA), Tim-Tipin (1.84 μM:0.92 μM Tim-Tipin) and Tim-Tipin-RPA (1.2 μM:0.6 μM Tim-Tipin-RPA) are used. After incubation with the DNA substrate, the protein complexes were cross-linked with glutaraldehyde, analyzed by EMSA and visualized as in (A).

Inline graphic — Binding of Tim-Tipin-RPA to ssDNA substrates. (A) EMSA using 60 and 14 nt 5′-FAM labeled ssDNA substrate. The protein-ssDNA complexes were visualized by fluorescence imaging (EMSA), immunoblot analysis using an anti-RPA70 and anti-Tipin primary antibody and Coomassie stain. In the presence of long excess ssDNA significant amounts of intact Tim-Tipin-RPA-ssDNA complex were detected as indicated by the left square. The presence of substoichiometric amounts of long ssDNA (lanes 4 and 5) and short ssDNA (lanes 6–10) lead to dissociation of the Tim-Tipin-RPA-ssDNA complex into DNA-free Tim-Tipin and DNA-bound RPA. The total ssDNA concentration was varied from 64, 32, 8 to 4 μM as indicated by (). The Tim-Tipin-RPA concentration was kept constant at 16 μM. R, RPA; T, Tim-Tipin; P, pentameric Tim-Tipin-RPA. Lanes 11–13 show the DNA-free proteins. (B) EMSA after cross-linking the complex. Double excess amounts of 60 nt 5′-FAM labeled ssDNA and RPA (3.12 μM:1.56 μM RPA), Tim-Tipin (1.84 μM:0.92 μM Tim-Tipin) and Tim-Tipin-RPA (1.2 μM:0.6 μM Tim-Tipin-RPA) are used. After incubation with the DNA substrate, the protein complexes were cross-linked with glutaraldehyde, analyzed by EMSA and visualized as in (A).