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The Decondensation factor 31 binds to mono-nucleosomes

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2 Mono-nucleosomes were assembled on the Cy5- labeled 601 positioning sequence according to Rhodes and Laskey, and used at a concentration of 0.13 µM in the MST experiments (Rhodes and Laskey, 1989). To this a serial dilution of the Df31 protein was added with starting concentration at 65 µM. The samples were filled into standard capillaries and incubated at 27 °C for 15 min, prior to the MST measurement. For the assay three independent measurements were performed. A clear binding curve for the Df31 protein with mono-nucleosomes could be detected. However, BSA as control protein showed no binding to mono-nucleosomes. The calculated K d from the measurements of the Df31 binding to mono-nucleosome was 3.72 ± 0.56 µM. As control we also measured the interaction of BSA with Cy5-labeled mono- nucleosomes. The control experiment was performed as described for the Df31. No interaction could be detected. Conclusion The study provides an example that MicroScale Thermophoresis is capable of measuring and detecting specific interactions between nucleosomes and their interacting proteins. Straightforward control experiments proof the specificity of the interaction. Experiments are easily setup and affinities can be determined in a timely manner. Material and Methods Assay conditions For the experiment Cy5-labeled mono- nucleosomes were used at the concentration of 0.13 µM. Unlabeled Df31 and BSA proteins were added in 1:1 dilutions beginning at 65 µM. Samples were prepared in a buffer containing 20 mM Tris-HCl pH 7.4, 1.5 mM MgCl 2 , 0.5 mM EGTA, 200 mM KCl, 10 % Glycerol and 0.1 (v/v) % NP-40. For the measurement the samples were filled into standard capillaries. Instrumentation The measurements were performed on a NanoTemper Monolith NT.115 instrument. The measurement was performed in standard capillaries at 30 % LED and 50 % MST power, Laser-On time was 30 sec, Laser-Off time 5 sec. References Langst, G., and Becker, P. B. (2001). ISWI induces nucleosome sliding on nicked DNA. Mol Cell 8, 1085-1092. Rhodes, D., and Laskey, R. A. (1989). Assembly of nucleosomes and chromatin in vitro. Methods Enzymol 170, 575-585. Schubert, T., Pusch, M. C., Diermeier, S., Benes, V., Kremmer, E., Imhof, A., and Langst, G. (2012). Df31 Protein and snoRNAs Maintain Accessible Higher-Order Structures of Chromatin. Mol Cell. van-Holde, K. E. (1989). Chromatin. Springer Verlag, 497. © 2013 NanoTemper Technologies GmbH

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