Technologies

In Regensburg, we have an impressive research infrastucture. This includes:

Microscopy

For fluoresence-based single-molecule investigations, we operate two advanced fluorescence microscopes that provide single-molecule resolution.
• Single-molecule total internal reflection (TIRF) microscope: this microscope allows the visulization of immbolized single photon emitters, e.g. fluorescently labelled proteins and nucleic acids. Most commonly, this setup is used to perform single-molecule fluorescence energy transfer (FRET) measurements with individual biomolecules or biomolecular complexes. This microscope is a home-build setup (Grohmann group).
• Time-resolved confocal fluorescence microscope with single molecule sensitivity: The MT200 microscope (Picoquant) provides the possibility to analyze a multitude of parameters down to the single molecule level using methods such as Fluorescence Lifetime Imaging (FLIM), FRET, PIE, FCS/FCCS, burst analysis.

Mass Spectrometry

The mass spectrometry platform provides technology and instrumentation for a range of state-of-the-art proteomics approaches. This includes shotgun proteomics, interactome analysis, quantitative proteomics workflows, which can be either stable-isotope labeling based (e.g. SILAC and iTRAQ) or label-free, and the analysis of posttranslational modifications. In addition, we perform targeted proteomics (Selected reaction monitoring) for absolute and relative quantification of proteins or posttranslational modifications. Currently, we operate an ultra-high-resolution Q-TOF (UHR-QTOF) instrument coupled to a nanoflow HPLC (Ultimate 3000 RSLC). A second LC-ESI-MS/MS system, consisting of a QTRAP4500 mass spectrometer coupled to a nanoflow HPLC, is dedicated to targeted quantification (SRM). Furthermore, we use a multidimensional HPLC for fractionation of peptides and proteins by strong cation exchange (SCX) or size exclusion (SEC) chromatography. For the analysis of mass spectrometry data we have several bioinformatics software packages (PEAKS, Scaffold, MaxQuant, Skyline, ProteinScape, Mascot Distiller) available.

NMR Spectroscopy

Nuclear Magnetic Resonance (NMR) spectroscopy is the method of choice to detect and quantify biologically important motions in a near cellular environment. Using dedicated experiments, we can probe motions ranging from the pico- to nanoseconds time-scale up to the seconds time-scale, with atomic resolution. In addition, we can monitor how the molecular motions are affected by the interaction with adaptor complexes.
Traditionally, the application of NMR spectroscopy has been limited to complexes below 25 kDa in molecular weight. Recent advances have, however, extended this molecular weight regime into the hundreds of kDa and in favorable cases over 1 MDa. This makes the large molecular machines amenable to detailed studies of molecular dynamics.
In Regensburg, 500, 600 and 800 MHz NMR machines are installed. These are equipped with cryogenically cooled probeheads and state-of-the-art NEO consoles.

Cryo-EM

The ‘resolution revolution’ in cryo-EM is characterized by ground-breaking improvements in Electron microscope design, the development of direct electron detectors and novel software tools, leading to the award of the 2017 Nobel Prize in Chemistry to three outstanding contributors. High-resolution reconstructions of large macromolecular particles can be obtained in close-to-native states, allowing us to understand the biochemical basis of protein-protein (or protein-nucleic acid) interactions, enzymatic functions and their regulation in astonishing detail. Sample preparation and computational processing pipelines have been established under the roof of the RCB and SFB 960 and a state-of-the-art cryo-electron microscope (Jeol ‘Cryo-ARM 200’) equipped with direct electron detector (Gatan ‘K2’) has been installed with strong DFG-support to RCB members.

Microscale thermophoresis

The determination of quantitative parameters on the binding affinities of molecular interactions, the determination of protein quality, stability and size, are fundamental to the biochemical characterization of active biomolecules. We own the largest Microscale Thermophoresis Facility worldwide and additional nanoDifferential Scanning Fluorometry (nanoDSF) and Dynamic Light Scattering (DLS) equipment to solve these questions.