Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins.
Fluorescence recovery after photobleaching (FRAP) has become a popular technique to investigate the behavior of proteins in living cells. Although the technique is relatively old, its application to studying endogenous intracellular proteins in living cells is relatively recent and is a consequence of the newly developed fluorescent protein-based living cell protein tags. This is particularly true for nuclear proteins, in which endogenous protein mobility has only recently been studied. Here we examine the experimental design and analysis of FRAP experiments. Mathematical modeling of FRAP data enables the experimentalist to extract information such as the association and dissociation constants, distribution of a protein between mobile and immobilized pools, and the effective diffusion coefficient of the molecule under study. As experimentalists begin to dissect the relative influence of protein domains within individual proteins, this approach will allow a quantitative assessment of the relative influences of different molecular interactions on the steady-state distribution and protein function in vivo.
Carrero
Gustavo
G
Department of Mathematical and Statistical Sciences, University of Alberta, Alberta, Canada.
McDonald
Darin
D
Crawford
Ellen
E
de Vries
Gerda
G
Hendzel
Michael J
MJ
D005347:Fibroblasts / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008962:Models, Theoretical; D009687:Nuclear Proteins / Q000378:metabolism*
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D049452:Green Fluorescent Proteins