Discrimination of Kinetic Models by a Combination of Microirradiation and Fluorescence Photobleaching.
Fluorescence recovery after photobleaching (FRAP) is an excellent tool to measure the chemical rate constants of fluorescently labeled proteins in living cells. Usually FRAP experiments are conducted with the protein concentrations being in a steady state, i.e., when the association and dissociation of the proteins are equilibrated. This is a strong limitation because situations in which rate constants change with time are of great scientific interest. In this study, we present an approach in which FRAP is used shortly after DNA damage introducing laser microirradiation, which results in the recruitment of the DNA clamp protein proliferating cell nuclear antigen (PCNA) to DNA lesions. We establish different kinetic models that are compatible with the observed PCNA recruitment data if FRAP is not used. By using FRAP at different time points during protein accumulation, we can not only exclude two out of three models, but we can also determine the rate constants with increased reliability. This study thus demonstrates the feasibility of using FRAP during protein recruitment and its application in the discrimination of possible kinetic models.
Institute for Condensed Matter Physics, Technische Universität Darmstadt, Darmstadt, Germany. Electronic address: lengert@fkp.tu-darmstadt.de.
Laurin
L
Lengert
Institute for Condensed Matter Physics, Technische Universität Darmstadt, Darmstadt, Germany.
Nicor
N
Lengert
Institute for Condensed Matter Physics, Technische Universität Darmstadt, Darmstadt, Germany.
Barbara
B
Drossel
Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany.
M Cristina
MC
Cardoso
Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany.
Britta
B
Muster
Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany.
Danny
D
Nowak
Department of Biology, Technische Universität Darmstadt, Darmstadt, Germany.
Alexander
A
Rapp
D004249:DNA Damage / Q000502:physiology* / Q000528:radiation effects*; D005240:Feasibility Studies; D005453:Fluorescence; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D007834:Lasers; D018613:Microscopy, Confocal / Q000379:methods; D008954:Models, Biological; D038761:Photobleaching; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*
D018809:Proliferating Cell Nuclear Antigen