biostudies-literatureSmith EMNIGMS NIH HHSPHS HHS66-75https://www.ebi.ac.uk/biostudies/studies/S-EPMC4119281biostudies-literatureUnknown107(1)Fluorescently labeled proteins that are found both in the cytoplasm and at the plasma membrane, such as peripheral membrane proteins, create stratified fluorescent layers that present a challenging environment for brightness studies with fluorescence fluctuation spectroscopy. The geometry of each layer along with fluorescence and brightness contributions from adjacent layers generates a convoluted raw brightness that conceals the underlying brightness of each individual layer. Because the brightness at a layer establishes the oligomeric state of the fluorescently labeled protein at said layer, we developed a method that connects the experimental raw brightness with the physical brightness at each layered compartment. The technique determines the oligomerization in each compartment from an Biophysical journalQuantifying protein-protein interactions of peripheral membrane proteins by fluorescence brightness analysis.PMC41192815T32A1083196R01 GM064589R01GM064589Mueller JDChen YMacdonald PJSmith EMfalseQuantifying protein-protein interactions of peripheral membrane proteins by fluorescence brightness analysis.Fluorescently labeled proteins that are found both in the cytoplasm and at the plasma membrane, such as peripheral membrane proteins, create stratified fluorescent layers that present a challenging environment for brightness studies with fluorescence fluctuation spectroscopy. The geometry of each layer along with fluorescence and brightness contributions from adjacent layers generates a convoluted raw brightness that conceals the underlying brightness of each individual layer. Because the brightness at a layer establishes the oligomeric state of the fluorescently labeled protein at said layer, we developed a method that connects the experimental raw brightness with the physical brightness at each layered compartment. The technique determines the oligomerization in each compartment from an 2014-01-01T00:00:00Z2014 Jul2025-04-19T04:17:45.341Z2019-03-27T01:33:15ZS-EPMC41192812498834210.1016/j.bpj.2014.04.055