Fluorescent labeling and modification of proteins
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REVIEW
Fluorescent labeling and modification of proteins Christopher P. Toseland
Received: 15 February 2013 / Accepted: 1 April 2013 / Published online: 13 April 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract This review provides an outline for fluorescent labeling of proteins. Fluorescent assays are very diverse providing the most sensitive and robust methods for observing biological processes. Here, different types of labels and methods of attachment are discussed in combination with their fluorescent properties. The advantages and disadvantages of these different methods are highlighted, allowing the careful selection for different applications, ranging from ensemble spectroscopy assays through to single-molecule measurements. Keywords Fluorescence . Single molecule . Fluorophores . Fluorescent proteins . Quantum dots
Introduction Fluorescence-based assays are crucial experimental tools which allow the detailed dissection of molecular mechanisms. The assays can be performed in real-time, in solution, with high-time resolution and sensitivity down to single molecules. This has allowed the measurement of protein interactions, enzymatic activity, conformation changes, localization of proteins, and the ability to see individual proteins moving in real time [2, 8, 20, 42, 51, 59]. Proteins have intrinsic fluorescence due to residues such as tryptophan. However, extrinsic labels provide far greater advantages for observing biological processes. These include more flexibility in choosing the location of the label and enhanced fluorescence properties suitable for a greater number of techniques. A fluorophore reports upon the protein it is attached to and its environment. In the simplest case, this allows proteins to be visualized or counted through photobleaching. C. P. Toseland (*) Institut für Zelluläre Physiologie and Center for NanoScience (CeNS), Physiologisches Institut, Ludwig Maximilians Universität, Munich 80336, Germany e-mail: [email protected]
More advanced assays can measure protein–protein interactions or conformation changes, by measuring intensity changes and fluorescence/Förster resonance energy transfer (FRET). With all of these approaches, ensemble bulk or single-molecule measurements are possible. Bulk measurements offer the greater flexibility with the least requirements in terms of fluorescence properties. Single-molecule measurements provide a greater sensitivity because the ensemble averaging can mask rare, or short-lived, states [31]. Before selecting a label, it is important to define the type of experiment to be performed: Single molecule or ensemble spectroscopic measurements? Monitoring the protein of interest or use a fluorescent biosensor? Measure a conformation change, a FRET signal or visualize a protein? Each category has specific requirements for the label (Table 1). However, there are some general features. Ideally, a fluorescent label should be small, bright, and stable, without any perturbation to the biological system. Furthermore, the label should be specific wi
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