Quantifying membrane protein oligomerization using two-dimensional fluorescence intensity fluctuation spectrometry

Abstract Current methods for determining membrane protein association in cells are either very time consuming, require complicated procedures, or lack the sensitivity needed to assess the effect of concentration or ligand binding on the observed oligomerization. To overcome these limitations, we provide a detailed protocol for quantifying the relative abundance and stability of oligomers of differing sizes using two-dimensional fluorescence intensity fluctuation (2D-FIF) spectrometry. The approach can be implemented using a standard laser scanning fluorescence microscope along with custom written software for image analysis. This method may be applied to evaluate the extent of oligomerization as a function of receptor concentration and is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest.