Observing protein interactions and their stoichiometry in living cells by brightness analysis of fluorescence fluctuation experiments.
A single fluorescently labeled protein generates a short burst of light whenever it passes through a tiny observation volume created within a biological cell. The average amplitude of the burst is related to the stoichiometry of the fluorescently labeled protein complex. Fluorescence fluctuation spectroscopy quantifies the burst amplitude by introducing the brightness parameter. Brightness provides a spectroscopic marker for observing protein interactions and their stoichiometry directly inside cells. Not all fluorescent proteins are suitable for brightness experiments. Here we discuss how brightness properties of the fluorophore influence brightness measurements and how to identify a well-behaved fluorescent protein. Protein interactions and stoichiometry are determined from a brightness titration. Experimental details of brightness titration measurements are described together with the necessary calibration and control experiments.
Chen
Yan
Y
School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, USA.
Johnson
Jolene
J
Macdonald
Patrick
P
Wu
Bin
B
Mueller
Joachim D
JD
D000818:Animals; D002460:Cell Line; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011506:Proteins