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Special methodical expertise

Investigation of cellular signal-transduction using for example siRNA based techniques
Methods to analyse transcriptional gene regulation such as Chromatin immunoprecipitation (ChIP)
Analysis of protein/protein interaction using methods such as a pull down assays
Imaging techniques based on confocal laser scanning microscopy or life cell imaging

Apart from classical biochemical methods to analyse protein expression or secondary modifications of proteins, more specialized methods are used to analyze the role of a defined protein for cellular signal transduction. In particular the use of so-called small interference RNA to specifically suppress the expression of a defined protein is a valuable method for investigating its role for signal-transmission. A second more specialised technique is the use of ChIP to analyze the binding of regulators of gene expression, also termed as transcription factors, to their specific DNA binding element. These binding sites are located in regions of the chromatin which control the expression of a specific gene. The ChIP assay allows on the one hand to monitor binding of a transcription factor to a known binding site within the chromatin but can be also used to identify new gene regions/binding sites that are recognized by the respective regulator. To analyse protein/protein interactions a specific protein is expressed and purified either as the whole protein or as defined fragments of the protein and immobilized on a surface of a specific particle/bead. This immobilized protein/protein-fragment is than exposed to e.g. a protein-extract derived from whole tissue or cell culture. If this extract comprises proteins that will specifically bind to the immobilized protein these proteins will not be removed when the extracts are removed and the particles are rinsed. The interacting protein will be subsequently determined using standard procedures. This technique allows to characterize the protein/protein interaction of two proteins and to identify the respective regions of a protein that are important for this direct interaction. Finally methods based on immune fluorescence allow to visualize proteins using antibodies that are directed against the protein of interest, which are labelled with a fluorescing dye. These dyes can be detected by their ability to emit fluorescing light if excited by a light of a specific wavelength. Using laser light to excite these dyes a high spatial resolution can be achieved, providing information on sub cellular localization of a protein. The fact that a variety of dyes are available which particularly differ with respect to the emitted light and the light required for excitation makes it possible to detect two and more specific proteins in one probe.

Figure Legend:
A: Double immunofluorescence staining of the non-structural HCV protein NS5A (green) and the cellular tyrosine kinase Src (red) in the hepatoma cell line Huh7 harbouring the HCV replicase complex. In this staining it becomes clearly visible that Src is mainly located at the membrane of a cell whereas the NS5A protein is located within the cytoplasm of cells. Nuclei are stained in blue