Labeling of native proteins with fluorescent RAFT polymer probes: application to the detection of a cell surface protein using flow cytometry

Duret, Damien; Haftek-Terreau, Zofia; Carretier, Matthieu; Berki, Thomas; Ladavière, Catherine; Monier, Karine; Bouvet, Philippe; Marvel, Jacqueline; Leverrier, Yann; Charreyre, Marie‐Thérèse; Favier, Arnaud

doi:10.1039/c7py02064c

Cited by 14 publications

(21 citation statements)

References 43 publications

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“…As we previously reported that such polymer probes bearing AF647 fluorophores (but without the Halo-ligand) were not cell permeant, 5 this study was conducted with HeLa S3 cells engineered 21 to express the HaloTag at the cell surface with a GPI-anchor (Halo-GPI cells) (see Methods). Expression of the HaloTag transgene in the Halo-GPI cells was either induced with 50 nM doxycycline (Dox) for 48 h or not induced (negative control) followed by incubation with two types of fluorescent probes.…”

Section: Resultsmentioning

confidence: 99%

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Advanced Fluorescent Polymer Probes for the Site-Specific Labeling of Proteins in Live Cells Using the HaloTag Technology

et al. 2019

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We report the site-specific and covalent bioconjugation of fluorescent polymer chains to proteins in live cells using the HaloTag technology. Polymer chains bearing a Halo-ligand precisely located at their α-chain-end were synthesized in a controlled manner owing to the RAFT polymerization process. They were labeled in lateral position by several organic fluorophores such as AlexaFluor 647. The resulting Halo-ligand polymer probe was finally shown to selectively recognize and label HaloTag proteins present at the membrane of live cells using confocal fluorescence microscopy. Such a polymer bioconjugation approach holds great promises for various applications ranging from cell imaging to cell surface functionalization.

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Section: Resultsmentioning

confidence: 99%

“…4c The polymer chain size can be controlled owing to the RAFT polymerization process. In addition, we have already shown that such polymer chains can be functionalized at their chain end to promote an oriented conjugation onto native proteins 5 or histidine-tagged proteins in vitro via a nitrilotriacetic acid (NTA) ligand. 6…”

Section: Introductionmentioning

confidence: 99%

Advanced Fluorescent Polymer Probes for the Site-Specific Labeling of Proteins in Live Cells Using the HaloTag Technology

et al. 2019

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“…These polymers were synthesized from the same PNAM precursor bearing a succinimidyl ester chain-end (noted 0 in Scheme 2) prepared following a previously described procedure. 10 MALDI-TOF MS was then used to prove the chain-end modification, especially by conducting negative-ion analyses with non-stabilized THF.…”

Section: Resultsmentioning

confidence: 99%

Taking Advantage of Oxidation to Characterize Thiol-Containing Polymer Chains by MALDI-TOF Mass Spectrometry

et al. 2020

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MALDI-TOF mass spectrometry analyses revealed the oxidation of thiol-containing polymer chain-ends during sample preparation using THF as solvent. In these conditions, the extent of oxidation was hardly reproducible, and led to various types of oxidized compounds. Preparing the samples at the last minute using commercial THF stabilized with an anti-oxidant led to more reproducible results, with the least oxidation. However, it is demonstrated herein that thiol oxidation can be advantageously taken into profit to further ascertain the presence of the thiol at the polymer chain-end. To force thiol oxidation we used THF without any anti-oxidant stabilizer, thus more prone to form peroxides. Thiol-containing polymer chains can thereby be indirectly evidenced by the formation of oxidation products such as chain-chain disulfide bonds and sulfonic acid chains-ends. More importantly, in these oxidizing conditions and in the negative mode, sulfonic acid-terminated polymer chains can be more sensitively detected than thiol ones (the low pKa of sulfonic acids facilitating their anionization in MALDI source). In conclusion, performing MALDI-TOF mass spectrometry analyses in oxidizing conditions, as complement to regular analyses, was found to be very useful for the chain-end identification of different thiol-containing polymer chains.

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“…A significant effort in the field was done to develop bright staining reagents by employing multimerization of smallmolecule fluorophores on non-fluorescent polymeric backbones such as polymethacrylate derivatives, [113] copolymers of Nacryloylmorpholine and N-acryloxysuccinimide (poly(NAM-co-NAS)), [114] and many others [115][116][117] (Figure 6, Table 6). The requirements to these dyes are often similar to those of π-conjugated polymers, e. g. high water-solubility, presence of bioconjugation [a] M n .…”

Section: Polymer-based Fluorophore Multimersmentioning

confidence: 99%

Fluorophore Multimerization as an Efficient Approach towards Bright Protein Labels

2021

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Cell analysis techniques like flow cytometry and fluorescence microscopy are widely used to explore cells in real-time and provide important insights into a variety of cellular processes. These techniques require bright and specific staining reagents to generate strong fluorescence signal and enable reliable readout, making the choice of fluorescent labels a key aspect of experimental design. Much research has been done in the field of fluorophore development to generate bright small-mole-cule dyes, fluorescent proteins, and emitting nanoparticles. However, it remains challenging to modify biomolecules with multiple emitters and avoid self-quenching of such fluorophores at the same time. Herein, we present advances in multimerization-based methods for the generation of fluorescent labels with high brightness and discuss their advantages and limitations in the context of flow cytometry and fluorescence microscopy applications.

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Labeling of native proteins with fluorescent RAFT polymer probes: application to the detection of a cell surface protein using flow cytometry

Abstract: Fluorescent RAFT polymer probes with an activated ester reactive end-group can be advantageously used to label native proteins.

Cited by 14 publications

References 43 publications

Advanced Fluorescent Polymer Probes for the Site-Specific Labeling of Proteins in Live Cells Using the HaloTag Technology

Advanced Fluorescent Polymer Probes for the Site-Specific Labeling of Proteins in Live Cells Using the HaloTag Technology

Taking Advantage of Oxidation to Characterize Thiol-Containing Polymer Chains by MALDI-TOF Mass Spectrometry

Fluorophore Multimerization as an Efficient Approach towards Bright Protein Labels

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