Probing Membrane Protein Association Using Concentration‐Dependent Number and Brightness

We introduce concentration‐dependent number and brightness (cdN&B), a fluorescence fluctuation technique that can be implemented on a standard confocal microscope and can report on the thermodynamics of membrane protein association in the native plasma membrane. It uses transient transfection to enable measurements of oligomer size as a function of receptor concentration over a broad range, yielding the association constant. We discuss artifacts in cdN&B that are concentration‐dependent and can distort the oligomerization curves, and we outline procedures that can correct for them. Using cdN&B, we characterize the association of neuropilin 1 (NRP1), a protein that plays a critical role in the development of the embryonic cardiovascular and nervous systems. We show that NRP1 associates into a tetramer in a concentration‐dependent manner, and we quantify the strength of the association. This work demonstrates the utility of cdN&B as a powerful tool in biophysical chemistry.