Application Notes

2 An EGFP tagged version of Df31 was used at a concentration of 1.4 µM in the experiments. A serial dilution of the core histones was added with starting concentration between 26-36 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 interaction of the Df31 protein and the core histones H3 and H4 could be detected. However, the core histones H2A and H2B showed no binding. The calculated K d from the measurements of the Df31 binding to H3 was 1.5 ± 0.14 µM and H4 was 12 ± 0.6 µM. As control we also measured the interaction of EGFP alone with our target histones H3 and H4. The experiment was performed as described for the Df31-EGFP. No interaction could be detected. Conclusion The study provides an example that MicroScale Thermophoresis is capable of measuring and detecting specific interactions between histones 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 EGFP tagged Df31 and EFGP control protein were used at the concentration of 1.4 µM. Unlabeled core histones H2A, H2B, H3 and H4 were added in 1:1 dilutions beginning at ranges between 26-36 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, 0.1 (v/v) % NP-40 and 200 ng/µl BSA. For the measurement the samples were filled into standard capillaries. Instrumentation The measurements were performed on a NanoTemper Monolith NT.115 instrument. The measurements were performed in standard capillaries at 20 % LED and 50 % MST power with Laser-On time 30 sec and Laser-Off time 5 sec. References Luger, K., and Richmond, T. J. (1998). The histone tails of the nucleosome. Curr Opin Genet Dev 8, 140-146. 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. Wolffe, A. P. (1997). Histones, nucleosomes and the roles of chromatin structure in transcriptional control. Biochem Soc Trans 25, 354-358. © 2013 NanoTemper Technologies GmbH

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