Abstract
Studying protein-protein and protein-lipid interactions in their native environment is highly desirable, yet, the heterogeneity and complexity of cellular systems limits the repertoire of experimental methods available. In cells, interactions are often taking place in confined microenvironments where factors such as avidity, hindered diffusion, reduced dimensionality, crowding etc. strongly influence the binding kinetics and therefore it can be problematic to equate binding affinities obtained by bulk in-solution methods (e.g., Fluorescence Polarization, Isothermal titration calorimetry, Microscale thermophoresis) with those occurring in real cellular environments. The Supported Cell Membrane Sheet method presented here, addresses these issues by allowing access to the inner leaflet of the apical plasma membrane. The method is a highly versatile, near-native platform for both qualitative and quantitative studies of protein-protein and protein-lipid interactions occurring directly in or on the plasma membrane.
Highlights
[Background] Proper evaluation of the binding strength between biomolecules is pivotal for understanding cellular pathways and for rational drug development
Interactions are often taking place in confined microenvironments where factors such as avidity, hindered diffusion, reduced dimensionality, crowding etc. strongly influence the binding kinetics and it can be problematic to equate binding affinities obtained by bulk insolution methods (e.g., Fluorescence Polarization, Isothermal titration calorimetry, Microscale thermophoresis) with those occurring in real cellular environments
The general understanding of both the dynamics and kinetics of biomolecular interactions often relies on experimental approaches where single protein domains of interest are isolated from their native cellular environment and tested under conditions that may deviate significantly from the specific cellular compartment in which they occur
Summary
[Background] Proper evaluation of the binding strength between biomolecules is pivotal for understanding cellular pathways and for rational drug development. This exposes the inner leaflet of the plasma membrane for binding of fluorescently labeled ligands which can be studied by microscopy (Figure 1). Poly-L-ornithine hydrobromide (Sigma-Aldrich, catalog number: P-8638) 36. D-MEM (Gibco, Sigma-Aldrich, catalog number: 11965-092) 39.
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