Fluorescent Labeling of SNAP-Tagged Proteins in Cells

Lukinavičius, Gražvydas; Reymond, Luc; Johnsson, Kai

doi:10.1007/978-1-4939-2272-7_7

Cited by 19 publications

(11 citation statements)

References 37 publications

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“…Cells were grown in liquid YE5S medium at 32° C to exponential phase (OD 595 between 0.4 and 0.6) then diluted into liquid EMM5S medium and grown for 12 to 24 hours at 25° C before labeling with SNAP fluorophore (Keppler et al, 2003; Lukinavicius et al, 2015; Stagge et al, 2013). As discussed previously (Lacy et al, 2017), the SNAP-substrate fluorophore does not accumulate in high amounts in yeast cells containing multidrug exporter genes and intact cell walls.…”

Section: Methodsmentioning

confidence: 99%

Single-molecule turnover dynamics of actin and membrane coat proteins in clathrin-mediated endocytosis

Lacy

Baddeley

Berro

2019

Preprint

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Actin is required for clathrin-mediated endocytosis (CME) in yeast. Experimental observations indicate that this actin assembly generates force to deform the membrane and overcome the cell’s high turgor pressure, but the precise molecular details remain unresolved. Based on previous models, we predicted that actin at endocytic sites continually polymerize and disassemble, turning over multiple times during an endocytic event. Here we applied single-molecule speckle tracking in live fission yeast to directly measure this predicted turnover within the CME assembly for the first time. In contrast with the overall ~20-sec lifetimes of actin and actin-associated proteins in endocytic patches, we detected single-molecule residence times around 1 to 2 sec, and high turnover rates of membrane-associated proteins in CME. Furthermore, we find heterogeneous behaviors in many proteins’ motions. These results indicate that rapid and continuous turnover is a key feature of the endocytic machinery and suggest revising quantitative models of force production.

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Section: Methodsmentioning

confidence: 99%

Single-molecule turnover dynamics of actin and membrane coat proteins in clathrin-mediated endocytosis

Lacy

Baddeley

Berro

2019

Preprint

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“…For this purpose, we employed the well-established SNAP-tag technology [18][19][20][21][22][23]. The SNAP-tag is a self-labeling protein-tag, which allows a rapid and simple labelling reaction with a variety of benzylguanine (BG)-modified dyes or fluorophores with various wavelengths [19][20][21][24][25][26]. In previous studies, we could demonstrate the successful secretion and production of recombinant AV in a mammalian expression system [8].…”

Section: Introductionmentioning

confidence: 99%

Using the SNAP-Tag technology to easily measure and demonstrate apoptotic changes in cancer and blood cells with different dyes

et al. 2020

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In vitro and ex vivo development of novel therapeutic agents requires reliable and accurate analyses of the cell conditions they were preclinical tested for, such as apoptosis. The detection of apoptotic cells by annexin V (AV) coupled to fluorophores has often shown limitations in the choice of the dye due to interference with other fluorescent-labeled cell markers. The SNAP-tag technology is an easy, rapid and versatile method for functionalization of proteins and was therefore used for labeling AV with various fluorophores. We generated the fusion protein AV-SNAP and analyzed its capacity for the specific display of apoptotic cells in various assays with therapeutic agents. AV-SNAP showed an efficient coupling reaction with five different fluorescent dyes. Two selected fluorophores were tested with suspension, adherent and peripheral blood cells, treated by heat-shock or apoptosis-inducing therapeutic agents. Flow cytometry analysis of apoptotic cells revealed a strong visualization using AV-SNAP coupled to these two fluorophores exemplary, which was comparable to a commercial AV-Assay-kit. The combination of the apoptosis-specific binding protein AV with the SNAP-tag provides a novel solid method to facilitate protein labeling using several, easy to change, fluorescent dyes at once. It avoids high costs and allows an ordinary exchange of dyes and easier use of other fluorescent-labeled cell markers, which is of high interest for the preclinical testing of therapeutic agents in e.g. cancer research.

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“…A consecutively developed labeling system consists of the SNAP‐tag and CLIP‐tag that bind chemical probes with O6‐benzylguanine and O2‐benzylcytosine derivatives, respectively . Similarly, Halo‐tag binds synthetic dyes having a chloroalkane linker . Furthermore, cell‐permeable fluorescent probes that bind His‐tag were recently developed .…”

Section: Live Cell Multiplexed Imagingmentioning

confidence: 99%

Multiplexed imaging of intracellular protein networks

2016

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Cellular functions emerge from the collective action of a large number of different proteins. Understanding how these protein networks operate requires monitoring their components in intact cells. Due to intercellular and intracellular molecular variability, it is important to monitor simultaneously multiple components at high spatiotemporal resolution. However, inherent trade-offs narrow the boundaries of achievable multiplexed imaging. Pushing these boundaries is essential for a better understanding of cellular processes. Here the motivations, challenges and approaches for multiplexed imaging of intracellular protein networks are discussed. V C 2016 International Society for Advancement of Cytometry

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Fluorescent Labeling of SNAP-Tagged Proteins in Cells

Cited by 19 publications

References 37 publications

Single-molecule turnover dynamics of actin and membrane coat proteins in clathrin-mediated endocytosis

Single-molecule turnover dynamics of actin and membrane coat proteins in clathrin-mediated endocytosis

Using the SNAP-Tag technology to easily measure and demonstrate apoptotic changes in cancer and blood cells with different dyes

Multiplexed imaging of intracellular protein networks

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