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Institute of Physiology
University of Freiburg
Hermann-Herder-Str. 7
79104 Freiburg
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Physiology I

Physiology II
Prof. Bernd Fakler
Tel. +49 761 203-5176
Fax +49 761 203-5191
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  • We apply various configurations of the patch-clamp technique (whole-cell, cell-attached, excised inside-out, outside-out) to native and genetically manipulated cells and subcellular compartments. They enable us to monitor protein function and protein-protein interactions at high-resolution.
  • We use a large spectrum of biochemical techniques to detect and quantify membrane proteins (mainly ion channels and receptors), their post-translational modifications and association with other proteins in complexes and protein networks.
  • Modern mass spectrometers coupled with liquid chromatography enable us to identify several hundreds of proteins from complex samples with high confidence and sequence coverage. In addition, they provide quantitative data that let us determine stability, specificity and stoichiometry of protein-protein interactions as well as absolute protein abundance.
  • Nuclear magnetic resonance spectroscopy (NMR) provides information on structure and dynamics of biological macromolecules at atomic resolution under near-physiological conditions. We use it to examine proteins participating in the nano-environment of membrane proteins with regard to their 3D structure, mobility and interactions.
  • Using innovative microsystems, we work to enhance resolution and throughput of electrical recording of ionic currents. We develop biohybrid sensing devices based on single biological nanopores in membrane microarrays and study the interaction of natural and synthetic polymers with pore-forming membrane proteins.
  • To understand how neurons collectively process information, we develop optogenetic tools as well as new technologies for recordings from neurons in vivo and imaging of cell activity using photon Ca2+ and functional approaches. With computational network models we gain information on the principles underlying information processing in complex neuronal circuits.

Membrane Proteomics

Membrane Proteomics StrategyFunctional proteomic analysis plays a central role in modern systems biology and aims at comprehensive identification and characterization of protein protein interactions, i. e. the assembly of proteins into complexes and networks as a general functional and structural organisation principle.

In the projects of our research group functional proteomics is primarily used to unravel and characterize components and mechanisms of fast signal transduction processes at membranes which includes constitutive as well as transient or dynamic (membrane) protein interactions.

For this purpose we use a number of biochemical techniques which have been systematically developed and standardized in our laboratory as part of a basal strategy:

  • solubilization, isolation and separation starting from native tissue
  • mass spectrometric identification and quantification
  • heterologous reconstitution and biochemical in-vitro characterization

This strategy has been designed to achieve a maximum degree of reliability, sensitivity, closeness to the physiological situation, and rather complete identification of the proteins involved.

Membrane protein solubilization

Biochemical analysis of membrane proteins generally requires the use of detergents to make target proteins and protein complexes soluble. The challenge is to find a reasonable balance between high solubilization efficiency and preservation of structural integrity of proteins and protein assemblies. We monitor these parameters using different assays (Western blot analysis, native gel electrophoresis, binding assays) with a range of buffer systems to finally determine the optimal conditions. In doing so we benefit from the know-how and ComplexioLyte buffers of our collaboration partner Logopharm.

Native gel electrophoresis

BN-PAGE Separation of Membrane Protein Complexes
BN-PAGE separation of membrane protein complexes with rat brain example

Separation of protein complexes by native gel electrophoresis, originally developed by Schaegger and coworkers as blue native PAGE (BNPage) for analysis of mitochondrial membrane protein complexes, is one of the few options for high resolution separation of large protein complexes. Separation is achieved electrophoretically in two dimensions: by molecular (complex) size (0.1-10 MDa) along a pore gradient gel under native conditions and, after successive denaturation of the complexes, in a second dimension by SDS-PAGE according to the molecular weight of the corresponding subunits. The size distribution of target complexes as visualized by Western blot or mass spectrometric analysis, which may also be manipulated by addition of target-specific antibodies (so-called shift assays) allows for qualitative as well as quantitative conclusions about composition and stability of protein complexes.

Affinity purification

Affinity Purification Quantification and Example
Quantitative evaluation of an affinity purification and example of a target protein with different antibodies

Target proteins are isolated together with their associated partners by affinity capture, namely by using specific antibodies. Despite their generally high specificity and affinity, antibodies also have a number of adverse properties that can lead to significant errors in interaction analyses. Therefore, we use different stringent controls, such as target knockout tissue, and compare the results from multiple antibodies directed against the same target protein (multi epitope approach). When combined with quantitative mass spectrometric analysis, also rare or dynamic interaction partners can be identified with low error rates.

For a more detailed biochemical in-vitro characterization of protein-protein interactions we use a number of specialized techniques in addition: Protein complexes are reconstituted by co-expression in heterologous systems, in-vitro co-translation or in binding assays. Stability and stoichiometry of protein-protein interactions are analyzed by antibody shift assays, co-sedimentation centrifugation, analytical ultracentrifugation, gel filtration, photometric binding assays and quantitative mass spectrometry.

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