Pulse-Chase in the Light Microscope (original) (raw)
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Labeling tetracysteine-tagged proteins with biarsenical dyes for live cell imaging
Cold Spring Harbor protocols, 2011
Correlation of real-time or time-lapse light microscopy (LM) with electron microscopy (EM) of cells can be performed with biarsenical dyes. These dyes fluorescently label tetracysteine-tagged proteins so that they can be imaged with LM and, upon fluorescent photoconversion of 3,3'-diaminobenzidine tetrahydrochloride (DAB), with EM as well. In the following protocol, cells expressing tetracysteine-tagged proteins are labeled for 1 h with biarsenical dyes. The volumes indicated are for a single 30-mm culture dish containing 2 mL of labeling medium. Scale the suggested volumes up or down depending upon the size of the culture dish used in the labeling. The same procedure can be adapted for longer labeling times by lowering the amount of dye used to 50-100 nM; however, the amount of the competing dithiol EDT is maintained at 10-20 μM. Longer labeling times often produce higher signal-to-noise ratios and cause less trauma to the treated cells prior to imaging.
Nature Biotechnology, 2005
Membrane-permeant biarsenical dyes such as FlAsH and ReAsH fluoresce upon binding to genetically encoded tetracysteine motifs expressed in living cells 1,2 , yet spontaneous nonspecific background staining can prevent detection of weakly expressed or dilute proteins 2,3 . If the affinity of the tetracysteine peptide could be increased, more stringent dithiol washes should increase the contrast between specific and nonspecific staining. Residues surrounding the tetracysteine motif were randomized and fused to GFP, retrovirally transduced into mammalian cells and iteratively sorted by fluorescence-activated cell sorting for high FRET from GFP to ReAsH in the presence of increasing concentrations of dithiol competitors. The selected sequences show higher fluorescence quantum yields and markedly improved dithiol resistance, culminating in a >20-fold increase in contrast. The selected tetracysteine sequences, HRWCCPGCCKTF and FLNCCPGCCMEP, maintain their enhanced properties as fusions to either terminus of GFP or directly to b-actin. These improved biarsenical-tetracysteine motifs should enable detection of a much broader spectrum of cellular proteins.
Journal of Fluorescence, 2007
Investigating the localisation of proteins within live cells via fluorescence microscopy typically involves the fusion of the protein of interest to a large fluorescent protein such as green fluorescent protein (GFP). Alternate fluorescent labelling technologies such as the fluorescent biarsenical dye molecules (e.g. FlAsH, ReAsH) are preferable to the use of large fusion proteins in many respects and allow greater flexibility in terms of the location of the labelling site. We assessed the ability of the FlAsH-derived biarsenical dye molecule Lumio™ to label a range of tetracysteine containing proteins within live cells and report that although in some circumstances Lumio is capable of positively detecting such proteins, the sensitivity and specificity of labelling is significantly reduced, making the Lumio-labelling system unsuitable for the detection of a wide range of protein within live cells.
Journal of the American Chemical Society, 2002
for site-specific fluorescent labeling of recombinant proteins in living cells. The sequence Cys-Cys-Xaa-Xaa-Cys-Cys, where Xaa is an noncysteine amino acid, is genetically fused to or inserted within the protein, where it can be specifically recognized by a membrane-permeant fluorescein derivative with two As(III) substituents, FlAsH, which fluoresces only after the arsenics bind to the cysteine thiols. We now report kinetics and dissociation constants (∼10-11 M) for FlAsH binding to model tetracysteine peptides. Affinities in vitro and detection limits in living cells are optimized with Xaa-Xaa) Pro-Gly, suggesting that the preferred peptide conformation is a hairpin rather than the previously proposed R-helix. Many analogues of FlAsH have been synthesized, including ReAsH, a resorufin derivative excitable at 590 nm and fluorescing in the red. Analogous biarsenicals enable affinity chromatography, fluorescence anisotropy measurements, and electron-microscopic localization of tetracysteine-tagged proteins.
New tools for in vivo fluorescence tagging
Current Opinion in Plant Biology, 2005
Engineering of fluorescent proteins continues to produce new tools for in vivo studies. The current selection contains brighter, monomeric, spectral variants that will facilitate multiplex imaging and FRET, and a collection of optical highlighter proteins that might replace photoactivatable-GFP. These new highlighter proteins, which include proteins that have photoswitchable fluorescence characteristics and a protein whose fluorescence can be repeatedly turned on and off, should simplify refined analyses of protein dynamics and kinetics. Fluorescent protein-based systems have also been developed to allow facile detection of protein-protein interactions in planta. In addition, new tags in the form of peptides that bind fluorescent ligands and quantum dots offer the prospect of overcoming some of the limitations of fluorescent proteins such as excessive size and insufficient brightness.