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Major Methods and Technical Approaches

With Fluorescence Correlation Spectroscopy (FCS) we study folding and unfolding of fluorescently labeled proteins. Depending on the concentration of denaturing chemicals, a change of the hydrodynamic radius is monitored by measuring diffusion times on the SM level.
Similarly, the efficiency of the Förster Resonance Energy Transfer (FRET) upon (un)folding allows to study conformational changes of multi-domain proteins in more detail. Proteins are site-specifically labeled with fluorescent dye pairs to sensitively study conformational protein changes and dynamics upon (un)folding and substrate binding. By SM multi-parameter analysis of our FRET data, we gain insight in conformational protein dynamics, state distributions and involved timescales.
Anisotropy experiments reveal the intrinsic dye mobility in order to determine dye-linker dynamics and to allow the entanglement of these motions from the overall protein dynamics.
SM imaging of ribosome-bound GFP allows studying co-translational folding via co-localization of the red-labeled ribosome and GFP fluorescence.

Zero-Mode-Waveguides (ZMWs)

The limited volume in such micro vessels allows working with higher concentrations than usually suitable for SM experiments. Additionally, field enhancement effects can be exploited to reduce the photo-stress on the sample.


zmw_bild_1


Greyscale image of an Zero-Mode-Waveguide Array [30μm X 30μm] with Atto655 labeled ribosome inside the holes when excited with a laser having the wavelengths of 640nm


Link to ICEMP-2014-International Conference on Emerging Materials and Processes and to one article of conference proceedings about Zero-Mode-Waveguides written by Dr. Jaydeep Bhattacharya.


Contact: Tina Züchner


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