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- W2052472175 abstract "Protein-protein interaction in live cell membranes can be shown by labelling the proteins of interest and mapping the distribution of the respective fluorescent signal in the membrane. Colocalization analysis of the images can indicate affinity of whatever kind, however background fluorescence, the excess of one interaction partner, coincidental overlaps and the difficulties of multicolor-imaging limit applicability and sensitivity of the approach. We presented a method to circumvent many of the difficulties by arranging one of the proteins as bait in a well-defined pattern in the cell membrane and measuring and comparing the abundance and diffusion characteristics of the second protein in bait-rich and bait-depleted areas and tested it on proteins of the immunological synapse as we showed the recruitment of Lck to CD4-enriched areas. To create those patterns the bait protein is immobilized by antibodies that are linked to Streptavidin, which is deposited in an underlying structure using soft lithography. The standard protocols for this technique allow only the creation of feature sizes which are at least one order of magnitude larger than the supposed size of functional domains in the cell membrane. To miniaturize the size of patterns to widths of 200nm and below great efforts in lithography are required as substrates have to be chosen more carefully and novel stamp materials such as polyhedral oligomeric silsesquioxane, which contain custom tailored functional groups, have to be used to achieve optimal stiffness control. The characterization of the imprints using combined AFM and fluorescence microscopy reveals a large environmental influence (humidity, temperature) during deposition of proteins and storage on the quality of the imprint and the substrate -protein binding affinity. We further investigate the micro- and nanoscale homogeneity of deposited streptavidin and demonstrate the usage of the miniaturized platform as a protein-protein interaction assay." @default.
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- W2052472175 date "2015-01-01" @default.
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- W2052472175 title "Pushing Micropatterning to the Nanoscale" @default.
- W2052472175 doi "https://doi.org/10.1016/j.bpj.2014.11.3431" @default.
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