Techniques and methodology

Protein biochemistry

Protein purification from bacterial systems. We clone and purify tagged (His, GST, MBP, Trx) and untagged proteins from E.coli using affinity chromatograpy and gelfiltration.

Protein purification mammalian systems. We clone and purify tagged (His, FLAG) and untagged proteins from HEK cells using affinity chromatograpy and gelfiltration.

General protein analysis. SDS-PAGE, Immunoblotting, gradient centrifugation, CD-spectroscopy,

Lipid biochemistry

Liposome-based model membrane systems. We generate small (50-500 nm) liposomes of defined lipid composition for liposome-based co-sedimentation assays and analysis of protein assembly on membranes by electron microscopy.

GUV-based model membranes. We generate giant unilamellar vesicles of defined lipid composition for microscopy-based analysis of protein assembly.

Supported bilayer-based model membranes. We generate supported bilayer membranes for microscopy-based analysis of protein assembly.

Fusogenic liposomes. We generate fusogenic liposomes of defined composition that are used for immidiate incorporation of lipids into the plasma membrane of cells.

Interaction studies

Protein-protein interactions. To meassure and assay protein interactions we use pull-down-based techniques, Isothermal titration caliriometry (ITC),Quarts crystal microbalance with dissipation monitoring (QCM-D), Immunoprecipitation and Fluorescence Life-time Imaging Microscopy (FLIM) for measurments in cells.

Protein-lipid interactions. To meassure and assay protein and lipid interactions we use liposome co-sedimentation assays, supported bilayers and Quarts crystal microbalance with dissipation monitoring (QCM-D), insertion of proteins in lipid monolayers using a Langmuir trough and microscopy-based assays of binding to model membranes.

Protein-protein interactions at the membrane interphase. We use FRET-based assays of reconstituted systems in vitro and FLIM or FRAP microscopy for studies in cells. We also analyse membrane bound compexes by electron microscopy.

Endocytic assays

Uptake up fluorescently labelled molecules. We couple small fluorescent molecules on target molecules (Dextran, proteins, lipids) and visualise internalisation by fluorometry or microscopy.

Single-particle tracking of the dynamics of endocytic carrier formation in cells. We use image analysis software to track the dynamics of fluorescent protein assemblying on the surface of living cells. We also track the trafficking of proteins and vesicles using spinning disc and TIRF microscopy.

siRNA-based knock down of proteins. To study the role of different proteins and uptake pathways in cells, we deplete the cells of key proteins using siRNA-based knock down.

Drugs to inhibit endocytosis or ohter processes. We use drugs to inhibit or slow down specific pathways.

Microinjection of proteins or antibodies.  We use a microinjector coupled to the confocal microscope to inject proteins that can be visualised.

Cellular models and organoid 3D-systems

FlpIn-TRex-HeLa cells. We design, clone and generate stable cell lines where 1-3 endocytic proteins have been tagged with fluorescent proteins for visualisation using microscopy. In these systems the level of expression is titrated to correspond to endogenous levels.

3T3-L1 cells. These cells are differentiated into adipocyte-like cells

Transient transfection. We have a library of fluorescently tagged proteins that can be transiently transfected and visualised using microscopy

MDCK 3D-model.

Light microscopy, atomic force microscopy and electron microscopy

Live cell confocal microscopy. Used for visualising proteins, lipids and organells in cells in real time.

Spinning disc microscopy with microfluidics. Used for visualisation of protein and lipid dynamics and rapid processes in cells.

Total internal reflection fluorescence microscopy (TIRF). Used for visualisation of processes taking place at the cell surface.

Fluorescence recovery after photobleaching microscopy  (FRAP). Used to analyse diffusion and binding of proteins and lipids in cells.

Fluorescent life time imaging microscopy (FLIM). Used to analyse and meassure interactions of proteins in living cells.

Correlative light and electron microscopy (CLEM). We use light microscopy to mark the area of interest and electron microscopy to provide high resolution images of the same area. We can combine light microscopy with cryoEM, TEM and SEM.

Correlative ligth and atomic force microscopy. We use light microscopy to mark the area of interest and AFM microscopy to provide high resolution measuments in the z-plane of the same area.

Transmission electron microscopy. We use negative stain electron microscopy to visualise protein assemblies on membranes.

Image analysis. We use image analysis softwares such as Imaris and Image J to quantify fluorescence and track single fluorescent particles over time.

Stucture biology

Cryo electron tomography. Used to determine the structure of protein assemblies on membranes.

Single particle cryo electron microscopy. Used to determine the structure of protein and lipid complexes.

CD spectroscopy. Used to analyse protein secondary structure.

Infrared Reflection Absorption Spectroscopy (IRRAS). We collaborate with Christian Schwieger in Halle for IRRAS.

Crystallography. We collaborate with experts in protein crystallography, Prof. Oliver Daumke, Berlin, Prof. Harvey McMahon, Cambridge.