Imaging proteins inside cells with fluorescent tags - PubMed (original) (raw)

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Imaging proteins inside cells with fluorescent tags

Georgeta Crivat et al. Trends Biotechnol. 2012 Jan.

Abstract

Watching biological molecules provides clues to their function and regulation. Some of the most powerful methods of labeling proteins for imaging use genetically encoded fluorescent fusion tags. There are four standard genetic methods of covalently tagging a protein with a fluorescent probe for cellular imaging. These use (i) autofluorescent proteins, (ii) self-labeling enzymes, (iii) enzymes that catalyze the attachment of a probe to a target sequence, and (iv) biarsenical dyes that target tetracysteine motifs. Each of these techniques has advantages and disadvantages. In this review, we cover new developments in these methods and discuss practical considerations for their use in imaging proteins inside living cells.

Published by Elsevier Ltd.

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Figures

Figure 1

Figure 1

Auto-fluorescent proteins as fusion tags. a) Table of excitation and emission wavelengths for twelve optimized fluorescent proteins (FP). The comparative brightness of each FP is listed as the product of the quantum yield and the extinction coefficient at the peak absorbance wavelength divided by 1000. Values were generating from the literature [1, 23, 26, 28]. b) Structure of EGFP [39]. A magnified view of the cyclized chromophore (TYG) is shown to the right. c) Cartoon of N- and C- terminal linkers introduced during genetic fusion of a protein of interest (POI) to an FP in standard Clontech vectors. The minimum and maximum linkers are indicated by parenthesis. The unstructured amino acids from EGFP are colored green. A common flexible peptide linker is shown below.

Figure 2

Figure 2

Self-labeling enzymes as fluorescent fusion tags. a) The structure of AGT/SNAP tag (top) and the Haloalkane dehydrogenase tag (bottom). The residues that are labeled by the fluorescent ligand are shown in yellow. In Halo, the mutated catalytic histidine is shown in orange. b) (top) Structure of a benzyl guanine-linked TMR ligand for SNAP lag labeling and a (bottom) TMR-linked haloalkane for Halotag labeling along with c) the corresponding reaction mechanisms for covalent attachment.

Figure 3

Figure 3

Structure of the biarsenical dyes FLAsH-EDT2 (a) and ReAsH-EDT2 (b). (c) Model of the optimized tetracysteine peptide bound to ReAsH based on the NMR structure of the complex [70].

Figure 4

Figure 4

Examples of fluorescent labeling methods in cells. a) Total Internal Reflection Fluorescence image (TIRF) of a living PC12 cells expressing the F-actin binding protein ITPKA tagged with tdTomato [85]. b) TIRF image of a PC12 cell expressing Halotag-Beta actin labeled with the red fluorophore TMR-halotag ligand. c) Confocal image of an erythrocyte infected with transgenic Plasmodium falciparum expressing the tetracysteine tag (TC)-containing protein KAHRP (+His)-TC labeled with ReAsH.

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