2
Fig. 1 Measurement of Df31 binding to nucleic acids. MST
values were normalized to fraction bound. 3 independent
measurements where performed to obtain the depicted binding
curve. The K
d
of the Df31-ssRNA interaction was
24.01 ± 3.15 µM.
The calculated K
d
from the measurements of the
Df31 binding to ssRNA was 24.01 ± 3.15 µM.
As control we also measured the interaction of
BSA and GST with our target ssRNA. The
experiment was performed as described for the
Df31 protein. No interaction could be detected.
Conclusion
The study provides an example that MicroScale
Thermophoresis is capable of measuring and
detecting specific interactions between nucleic
acids 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 random FAM or Cy5-labeled
ssRNA, ssDNA and dsDNA molecules of same
sequence and length were used at the
concentration of 50 nM. Unlabeled Df31 was
added in 1:1 dilutions beginning at 680 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
0.05 (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 30 % 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