( Paul Gavin* Mark Prescott...)Minimizing Photobleaching of Blue Fluorescent Protein (BFP) using ,,, the Varian Cary Eclipse Fluorescence Spectrophotometer

Paul Gavin*, Mark Prescott*, Ph.D, Daren J. Fyfe#, Ph.D and Jeffrey J. Comerford#, Ph.D

* Department of Biochemistry and Molecular Biology,
Monash University,
Clayton campus,
Victoria 3800, Australia

# Technical Assistance,
Varian Australia,
679 Springvale Road,
Mulgrave,
Victoria 3170, Australia
E-mail: fluorescence@varianinc.com

Introduction

The term photobleaching refers to loss of fluorescence emission from samples due to prolonged exposure to exciting radiation. While many fluorophores retain stable fluorescence after extended periods of illumination, some photobleach after short periods of time.

In order to maintain optimal fluorescence emission, it is important to minimize photobleaching. Strategies to achieve this usually involve limiting one or both of the following: (1) the time of exposure to, or (2) the intensity of, the exciting light. However, either of these strategies may compromise the quality of the results or limit the types of analyses that can be performed because the signal to noise ratio (S/N) is unavoidably decreased. Furthermore, kinetics-based assays performed over an extended period of time may not be possible due to increased exposure of the fluorophore to the excitation light which results in photobleaching.

Blue Fluorescent Protein (BFP) provides an example of a fluorophore that is susceptible to photobleaching: even with folding improvements, BFP still suffers from a relatively low fluorescence quantum yield [compared to green fluorescent protein] and relatively easy bleaching. (Tsien, 1998)¹. Despite the use
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