Poster Presentations
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A New Approach in Systems Biology: Analysis and Quantification of Signalling Molecules with Reverse Phase Protein Microarrays
Robert Wellhausen, PhD Student, Max Planck Institute for molecular Genetics
The quantitative, time resolved monitoring of proteins, their modifications and local distribution, serves as a measurement for their activity. Reverse phase protein microarrays are a method allowing relative protein quantification. These arrays offer the potential to analyze small quantity samples, at several time points, with different sets of experiments and additionally several replicates at once. This technique was used to generate datasets allowing the quantification of proteins and their state (post translation modification) in cells upon external stimuli. This platform is able to detect changes in post translational modifications even in low copy proteins like the transcription factor CREB. Optimization of the working protocol is the key to reproducible and reliable data and improves the sensitivity of the assay. Equimolar protein concentrations guarantee comparability between different samples. Whereas several replicates eliminate false positive results and improve reproducibility. To study time dependent phosphorylation a model system applied to the PKA pathway, the isoprenaline stimulation of cells, resulted in time dependant CREB S133 phosphorylation. Therefore several nanoliters of cell lysate were spotted onto epoxy coated slides and probed with specific antibodies. The phosphorylation time series shown in western blots correlates with the one seen in array experiments.
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