A new multiplexing high throughput protein-protein-proximity assay for probing nano-scale protein organization and interaction in biological membrane.

Dr. Jianying Yang (Institute of Biology III, University of Freiburg)

The concept of plasma membrane organization has recently undergone a paradigm shift from the continuum fluid to the partitioned fluid model. Correspondingly, cell surface receptors are found to be non-randomly organized at nano-scale distances in the plasma membrane. The relocalization of these proteins is thus likely to be a crucial event for cell activation. In the past decades, a lot of methods including blue native polyacrylamide gel electrophoresis (BN-PAGE), Förster resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), single particle tracking (SPT), and super-resolution microscopy have been developed to study the nanoscale membrane protein organization. Knowledge accumulated from studies using these methods has contributed enormously to our understanding of membrane protein and lipid organization. However, a method that can simultaneously probe the nanoscale organization of several different kinds of receptors and yet still provide single cell resolution and fulfill the requirement to be high throughput is still missing and necessary for our studying.

To deal with this problem, we will develop a novel multiplexing high throughput protein-protein-proximity assay for probing nano-scale protein organization and interaction in biological membrane by combining several cutting-edge techniques and we will use the B cell antigen receptor that dissociate to initiate its signaling as a modeling system to evaluate and improve this new assay.