Protein Micropatterning in the Plasma Membrane Allows for Kd Determination in Living Cells and Superresolution Analysis of Lipid Rafts

Abstract

In 2008 we introduced an assay for quantitative analysis of the interaction between a fluorescently marked protein (prey) and a membrane protein (bait) using microstructured surfaces, produced by μ-contact printing, covered with ligands (antibodies) targeted against the bait. The proof-of-concept was demonstrated for the interaction between CD4, a major co-receptor in T cell signalling, and Lck, a protein tyrosine kinase essential for early T cell signalling (Schwarzenbacher, et al., Nat Methods, 2008). Since then we worked on technical advances of the assay which are presented here. A whole binding curve of secondary antibodies binding to immobilized mouse antibodies was recorded, demonstrating that our assay allows for determination of the dissociation constant between a bait and a prey protein in living cells. Furthermore we show that our method is a powerful tool to study membrane micro domains or lipid rafts: Via patterning of GPI-anchored GFPs the structure of cells membrane was some kind of sorted and the behaviour of various proteins in these regions have been analyzed by super resolution microscopy.