Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

Zanetti-Domingues, Laura C.; Tynan, Christopher J.; Rolfe, Daniel J.; Clarke, David T.; Martin-Fernandez, Marisa L.

doi:10.1371/journal.pone.0074200

Cited by 163 publications

(171 citation statements)

References 34 publications

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“…The Boltzmann function was fitted to each curve, and the pH at the half-point of absorbance (where half of the free dye molecules exist as [A - ] and the other half as [HA]) is defined to be the pK a . The measured pK a is consistent with the net charge of each dye, as can be seen in the case of Alexa 488, which has a net charge of 3.94 at pH 7.4 and showed the lowest pK a of 3.1 (6). The pK a values of free Cy5 and DNA were measured as 4.8 and 1.6, respectively, so we can achieve purification for Cy5-labeled DNA with an aqueous solution of pH 3.0.…”

supporting

confidence: 82%

General and Facile Purification of Dye-Labeled Oligonucleotides by Ph-Controlled Extraction

et al. 2018

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Previously, we reported a method for facile purification of oligonucleotides labeled with hydrophobic dyes, based on the solubility difference between the hydrophilic DNA and unreacted dye. Here, we present a new purification method applicable to any dye regardless of its hydrophobicity. We exploited the population shift of a fluorescent dye in a low-pH aqueous solution from its anionic form toward its neutral form. When the pH of an aqueous solution containing dye-labeled DNA and unreacted free dye is lowered, and the solution is mixed with a hydrophobic organic solvent (butanol), the neutral free dye is preferentially dissolved in the organic phase, leaving behind the hydrophilic dye-labeled DNA in the aqueous phase. We experimentally verified that our new method results in high yields of dye-labeled oligonucleotides and the efficient removal of free dye.

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supporting

confidence: 82%

General and Facile Purification of Dye-Labeled Oligonucleotides by Ph-Controlled Extraction

et al. 2018

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“…However, care has to be taken regarding the hydrophobicity of the dyes. Especially Atto647N is known for its hydrophobicity 53 , which can lead to nonspecific interactions with other molecules or even the labeled protein itself. It is therefore essential to characterize labeled proteins in vitro before using them in in vivo experiments: for example, regarding the conformational stability (Fig.…”

Section: Methodsmentioning

confidence: 99%

Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells

et al. 2015

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Single-molecule methods have become widely used for quantifying the conformational heterogeneity and structural dynamics of biomolecules in vitro. Their application in vivo, however, has remained challenging owing to shortcomings in the design and reproducible delivery of labeled molecules, the range of applicable analysis methods, and suboptimal cell culture conditions. By addressing these limitations in an integrated approach, we demonstrate the feasibility of probing protein dynamics from milliseconds down to the nanosecond regime in live eukaryotic cells with confocal single-molecule Förster resonance energy transfer (FRET) spectroscopy. We illustrate the versatility of the approach by determining the dimensions and submicrosecond chain dynamics of an intrinsically disordered protein; by detecting even subtle changes in the temperature dependence of protein stability, including in-cell cold denaturation; and by quantifying the folding dynamics of a small protein. The methodology opens possibilities for assessing the effect of the cellular environment on biomolecular conformation, dynamics and function.

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“…in RBC haemoglobin close to the plasma membrane could be a factor influencing alexa fluor ® 647. Isomerisation of double bonds of cyanine dyes is well known [45,46]. Such changes in combination with wavelength dependencies of the detectors may account, at least partly, for the differences observed for PS detection based on FITC and alexa fluor ® 647.…”

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confidence: 99%

Measurements of Intracellular Ca2+ Content and Phosphatidylserine Exposure in Human Red Blood Cells: Methodological Issues

Wesseling

Wagner-Britz²,

Boukhdoud³

et al. 2016

Cell Physiol Biochem

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Background/Aims: The increase of the intracellular Ca²⁺ content as well as the exposure of phosphatidylserine (PS) on the outer cell membrane surface after activation of red blood cells (RBCs) by lysophosphatidic acid (LPA) has been investigated by a variety of research groups. Carrying out experiments, which we described in several previous publications, we observed some discrepancies when comparing data obtained by different investigators within our research group and also between batches of LPA. In addition, we found differences comparing the results of double and single labelling experiments (for Ca²⁺ and PS). Furthermore, the results of PS exposure depended on the fluorescent dye used (annexin V-FITC versus annexin V alexa fluor® 647). Therefore, it seems necessary to investigate these methodological approaches in more detail to be able to quantify results and to compare data obtained by different research groups. Methods: The intracellular Ca²⁺ content and the PS exposure of RBCs separated from whole blood have been investigated after treatment with LPA (2.5 µM) obtained from three different companies (Sigma-Aldrich, Cayman Chemical Company, and Santa Cruz Biotechnology Inc.). Fluo-4 and x-rhod-1 have been used to detect intracellular Ca²⁺ content, annexin V alexa fluor® 647 and annexin V-FITC have been used for PS exposure measurements. Both parameters (Ca²⁺ content, PS exposure) were studied using flow cytometry and fluorescence microscopy. Results: The percentage of RBCs showing increased intracellular Ca²⁺ content as well as PS exposure changes significantly between different LPA manufacturers as well as on the condition of mixing of LPA with the RBC suspension. Furthermore, the percentage of RBCs showing PS exposure is reduced in double labelling compared to single labelling experiments and depends also on the fluorescent dye used. Finally, data on Ca²⁺ content are slightly affected whereas PS exposure data are not affected significantly by the measuring method (flow cytometry, fluorescence microscopy). Conclusion: The LPA batch used and the mixing procedure of LPA and the RBC suspension has to be taken into consideration when comparing results of intracellular Ca²⁺ content and PS exposure of RBCs after LPA activation. In addition, one should consider that the results of single and double labelling experiments might be different depending on the fluorescent dyes used.

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Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

Cited by 163 publications

References 34 publications

General and Facile Purification of Dye-Labeled Oligonucleotides by Ph-Controlled Extraction

General and Facile Purification of Dye-Labeled Oligonucleotides by Ph-Controlled Extraction

Single-molecule spectroscopy of protein conformational dynamics in live eukaryotic cells

Measurements of Intracellular Ca2+ Content and Phosphatidylserine Exposure in Human Red Blood Cells: Methodological Issues

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