Calibration-free in vitro quantification of protein homo-oligomerization using commercial instrumentation and free, open source brightness analysis software
JoVE. 2018-07-17; (137):
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1. J Vis Exp. 2018 Jul 17;(137). doi: 10.3791/58157.
Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using
Commercial Instrumentation and Free, Open Source Brightness Analysis Software.
Nolan R(1), Alvarez LA(1), Griffiths SC(2), Elegheert J(2), Siebold C(2),
(1)Cellular Imaging Group, Wellcome Centre Human Genetics, University of Oxford.
(2)Division of Structural Biology, Wellcome Centre Human Genetics, University of
(3)Cellular Imaging Group, Wellcome Centre Human Genetics, University of Oxford;
Division of Structural Biology, Wellcome Centre Human Genetics, University of
Oxford; Dynamic Structural Virology Group, Biocruces Health Research Centre;
IKERBASQUE, Basque Foundation for Science; .
Number and brightness is a calibration-free fluorescence fluctuation spectroscopy
(FFS) technique for detecting protein homo-oligomerization. It can be employed
using a conventional confocal microscope equipped with digital detectors. A
protocol for the use of the technique in vitro is shown by means of a use case
where number and brightness can be seen to accurately quantify the oligomeric
state of mVenus-labelled FKBP12F36V before and after the addition of the
dimerizing drug AP20187. The importance of using the correct microscope
acquisition parameters and the correct data preprocessing and analysis methods
are discussed. In particular, the importance of the choice of photobleaching
correction is stressed. This inexpensive method can be employed to study
protein-protein interactions in many biological contexts.
PMCID: PMC6126508 [Available on 2020-07-17]
PMID: 30080195 [Indexed for MEDLINE]