Diffusion‐based size determination of solute particles: a method adapted for postsynaptic proteins

The postsynaptic density (PSD) is a complex, multilayered protein network largely situated on the internal surface of the postsynaptic membrane. It is the first processing unit for incoming synaptic signals, and changes in its internal structure are associated with synaptic strength and plasticity. These structural changes are largely governed by multivalent interactions between its components. The in vitro characterization of such complexes requires unbiased methods that can be used to estimate the size of the emerging assemblies for systems with multiple possible stoichiometries. Here, we present an experimental method for detecting specific PSD proteins as well as their complexes based on their diffusion in a microfluidic environment. The method requires a fluorescent labeling technique that does not disrupt the function of labeled proteins, a microfluidic device that can maintain laminar flow for protein solutions, a microscope that can record the fluorescent signal emitted by these solutions, and an analytic software package that can process the collected experimental data and convert them into approximate particle sizes. We demonstrate the applicability of our method on protein constructs of various postsynaptic proteins, including the multivalent assembly between GKAP and LC8.