Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein–protein and protein–nucleic acid interactions

Rusinova, E. V.; Tretyachenko-Ladokhina, Vira; Vele, Oana; Senear, Donald F.; Ross, J. B. Alexander

doi:10.1016/s0003-2697(02)00325-1

Cited by 80 publications

(75 citation statements)

References 16 publications

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“…Fluorescence polarization was measured on a Tecan Ultra plate reader (Durham, NC) with excitation at 485 nm and emission at 535 nm. For each condition, polarization was calculated using the formula (I h Ϫ I v )/(I h ϩ I v ), where I h represents intensity measured with horizontally polarized excitation, and I v represents intensity measured with vertically polarized excitation and expressed as millipolarization (40).…”

Section: Analysis Of Proteins In Gastricmentioning

confidence: 99%

Helicobacter pylori Induces ERK-dependent Formation of a Phospho-c-Fos·c-Jun Activator Protein-1 Complex That Causes Apoptosis in Macrophages

Asim

Chaturvedi

Hoge

et al. 2010

Journal of Biological Chemistry

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Macrophages are essential components of innate immunity, and apoptosis of these cells impairs mucosal defense to microbes. Helicobacter pylori is a gastric pathogen that infects half of the world population and causes peptic ulcer disease and gastric cancer. The host inflammatory response fails to eradicate the organism. We have reported that H. pylori induces apoptosis of macrophages by generation of polyamines from ornithine decarboxylase (

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Section: Analysis Of Proteins In Gastricmentioning

confidence: 99%

Helicobacter pylori Induces ERK-dependent Formation of a Phospho-c-Fos·c-Jun Activator Protein-1 Complex That Causes Apoptosis in Macrophages

Asim

Chaturvedi

Hoge

et al. 2010

Journal of Biological Chemistry

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“…There have been comparative studies on the feasibility of several dyes for fluorescence anisotropy. Alexa and Oregon dyes conjugates with biological molecules (e.g., lipids), for instance, were found to be suitable for this method (Rusinova et al 2002). Additionally, NBD and DHP lipid conjugates were used for fluorescence anisotropy to detect rafts in living cells (Gidwani et al 2001).…”

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confidence: 99%

Fluorescence Techniques to Study Lipid Dynamics

Sezgin¹,

Schwille²

2011

Cold Spring Harbor Perspectives in Biology

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Biological research has always tremendously benefited from the development of key methodology. In fact, it was the advent of microscopy that shaped our understanding of cells as the fundamental units of life. Microscopic techniques are still central to the elucidation of biological units and processes, but equally important are methods that allow access to the dimension of time, to investigate the dynamics of molecular functions and interactions. Here, fluorescence spectroscopy with its sensitivity to access the single-molecule level, and its large temporal resolution, has been opening up fully new perspectives for cell biology. Here we summarize the key fluorescent techniques used to study cellular dynamics, with the focus on lipid and membrane systems.T o elucidate cellular processes in their native dynamic environment has been one of the main issues in cell biology over the past decades. The lack of appropriate techniques has long been the main limiting step for the research on dynamic systems, because it was impossible to acquire real time information with the well-known biochemical techniques. The key challenge in dynamically observing biological systems is to combine the ability to resolve moderate to very low concentrations of molecules-because they are simply limited in living cells-on relevant timescales. Relevant timescales in cell biology can be minutes and hours, on a systemic level of cell metabolism, down to the microsecond and even nanosecond regime in which molecular and intramolecular rearrangements take place. With respect to lipidic systems, relevant dynamics range from the local movements of lipids by diffusion to the mechanical transformations of whole membranes, spanning several orders of magnitude in time to be covered. Like for other cellular processes, the investigation of lipids and membranes also in general benefited greatly from the introduction of fluorescence microscopy and spectroscopy to biology. After the 1960s, great technological inventions based on the phenomenon of fluorescence were made, such as confocal microscopy, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), total internal reflection fluorescence (TIRF), and two-photon microscopy, that not only revolutionized imaging but also yielded access to dynamics on previously inaccessible timescales. Another very big step was certainly taken after the introduction of fluorescent proteins, which again accelerated the use of these techniques in living cells and organisms. Nowadays, the technical advancements of fluorescence-based methods allow us to explore systems as small as single molecules with temporal resolution down to the nanoseconds regime. Lately, even the resolution limit of optical microscopy, for a long time being one of the fundamental barriers in elucidating cellular processes, has been overcome by smart applications of the phenomenon of fluorescence.This article aims at giving a short overview on mainly fluorescence-based metho...

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“…5 Further, since OG has nearly the same structure, molecular weight, and emission and excitation characteristics as NaF (Table 1), efficient utilization of existing equipment and research protocols should be feasible.…”

Section: Introductionmentioning

confidence: 99%

Trans-Scleral Permeability of Oregon Green 488®

Lee

Kim²,

Kim³

et al. 2008

Journal of Ocular Pharmacology and Therapeutics

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Purpose: The aim of this study was to determine the scleral permeability of a commercially available version of 2Ј,7Ј-difluorofluorescein (OG) and compare it to that of sodium fluorescein (NaF). Methods: Both in vitro and in vivo experiments were performed. For the ex vivo experiment, a Lucite block perfusion chamber with human donor sclera was used. Two hundred microliters (200 L) of 2.5 mg/ml OG or NaF was placed in the donor chamber. The OG and NaF concentration that diffused across the sclera was measured every 2 h for 24 h by fluorometry, and the fluorescence in the sclera was examined by fluorescent microscopy. In vivo experiments consisted of live rabbits treated with a 0.2-mL subtenon injection of 7.5 mg/ml solution of either OG or NaF in the right eye. Intraocular fluorescence was measured by ocular fluorophotometry. Results: The scleral permeability coefficient (K trans ) of OG was 3.93 Ϯ 1.01 ϫ 10 Ϫ7 cm/sec and that of NaF was 4.41 Ϯ 1.32 ϫ 10 Ϫ7 cm/s. Both OG and NaF were visible throughout the sclera after 24 hours. Peak vitreous concentration after subtenon injection in rabbits was 6.48 Ϯ 2.65 ng/mL of OG at 2 min and 47.15 Ϯ 13.3 ng/mL of NaF at 10 min. Conclusions: OG was able to diffuse across the sclera and thus could be potentially useful as a fluorescent tag for intraocular drug delivery studies. However, its permeability was substantially less than that of NaF. 579

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Alexa and Oregon Green dyes as fluorescence anisotropy probes for measuring protein–protein and protein–nucleic acid interactions

Cited by 80 publications

References 16 publications

Helicobacter pylori Induces ERK-dependent Formation of a Phospho-c-Fos·c-Jun Activator Protein-1 Complex That Causes Apoptosis in Macrophages

Helicobacter pylori Induces ERK-dependent Formation of a Phospho-c-Fos·c-Jun Activator Protein-1 Complex That Causes Apoptosis in Macrophages

Fluorescence Techniques to Study Lipid Dynamics

Trans-Scleral Permeability of Oregon Green 488®

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