Protein Micropatterning Assay: Quantitative Analysis of Protein–Protein Interactions
Characterization, especially quantification, of protein interactions in live cells is usually not an easy endeavor. Here, we describe a straightforward method to identify and quantify the interaction of a membrane protein (“bait”) and a fluorescently labeled interaction partner (“prey”) (membrane-bound or cytosolic) in live cells using Total Internal Reflection Fluorescence microscopy. The bait protein is immobilized within patterns in the plasma membrane (e.g., via an antibody); the bait–prey interaction strength can be quantified by determining the prey bulk fluorescence intensity with respect to the bait patterns. This method is particularly suitable also for the analysis of weak, transient interactions that are not easily accessible with other methods.
Key wordsMicropatterning Protein–protein interactions Soft lithography TIRF microscopy Quantitive analysis Membrane proteins
This work was funded by the Austrian Science Fund (FWF projects P 26337 and P 25730), the Austrian Research Promotion Agency (FFG project 842379), the program ‘Regionale Wettbewerbsfähigkeit OÖ 2007–2013’ with the financial support of the European Fund for Regional Development, as well as the Federal State of Upper Austria.
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