Detection of Epitope-Tagged Proteins in Flow Cytometry: Fluorescence Resonance Energy Transfer-Based Assays on Beads with Femtomole Resolution

Buranda, Tione; López, Gabriel P.; Simons, Peter C.; Pastuszyn, Andrzej; Sklar, Larry A.

doi:10.1006/abio.2001.5363

Cited by 32 publications

(65 citation statements)

References 19 publications

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“…Quantum ™ FITC beads are based on the properties of a NIST-calibrated fluorescein solution. Thus in order to be used accurately, the end user must have to account for the inevitable changes in quantum yields and spectral mismatches that occur when fluorescein is functionalized into a fluorescent tag (21,26).…”

Section: Resultsmentioning

confidence: 99%

“…Phosphate-buffered saline (PBS) was purchased from Mediatech, Inc, Herndon, VA). Biotinylated FLAG (FLAG bio ) and FITC conjugated FLAG peptides (FLAG FITC ) were synthesized at UNM as described elsewhere (21). TRIS (10 mM or 25 mM Tris, 150 mM NaCl, pH7.5) and HHB (30 mM HEPES, 110 mM NaCl, 10 mM KCl, 1 mM MgCl 2 6H 2 O and 10 mM glucose, pH7.4) buffer were used in the presence or absence of 0.1% bovine serum albumim (BSA).…”

Section: Methodsmentioning

confidence: 99%

“…Biotinylated M2 anti-FLAG antibody coated streptavidin beads (M2 beads) were prepared as previously described (21). Commercially available biotinylated M2 antibodies have about 3-4 biotin conjugates on the Fc domain of the antibody according to manufacturer's data sheet.…”

Section: Preparation Of Qdot-labeled Microbeadsmentioning

confidence: 99%

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The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

Campos

López

et al. 2007

Analytical Biochemistry

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The use of fluorescence calibration beads has been the hallmark of quantitative flow cytometry. It has enabled the direct comparison of inter-laboratory data as well as quality control in clinical flow cytometry. In this paper we have described a simple method for producing color-generalizable calibration beads based on streptavidin functionalized quantum dots. Based on their broad absorption spectra and relatively narrow emission, that is tunable on the basis of dot-size, quantum dot calibration beads can be made for any fluorophore that matches their emission color. In an earlier publication (1) we characterized the spectroscopic properties of commercial streptavidin functionalized dots (Invitrogen). Here we describe the molecular assembly of these dots on biotinylated beads. The law of mass action is used to readily define the site densities of the dots on the beads. The applicability of these beads is tested against the industry standard, commercial fluorescein calibration beads. The utility of the calibration beads are also herein extended to the characterization surface densities of dot-labeled epidermal growth factor ligands as well as quantitative indicators of the binding of dotlabeled virus particles to cells.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

Campos

López

et al. 2007

Analytical Biochemistry

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“…Proteins were labeled with the desired probe as described previously [22]. The samples were purified and concentrated by ultra filtration from phosphate buffered saline using YM-30 Microcon centrifugal filter devices (Millipore Corp. Bedford, MA).…”

Section: Fluorescent Labeling Of α I3 Subunitsmentioning

confidence: 99%

“…G protein βγ dimers were prepared and purified as previously described [20,23]. Beads coated with ≈ 4 million M2 antiFLAG antibodies were prepared as previously described [22]. The M2 beads were resuspended in PBS buffer and stored in 25µL aliquots (40,000 beads/µL) at 4°C.…”

Section: Fluorescent Labeling Of α I3 Subunitsmentioning

confidence: 99%

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides

Buranda

Simons

et al. 2007

Analytical Biochemistry

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We have used rapid mix flow cytometry to analyze the early subsecond dynamics of the disassembly of ternary complexes of G-protein coupled receptors (GPCRs) immobilized on beads to examine individual steps associated with guanine nucleotide activation. Our earlier studies suggested that the slow dissociation of Gα and Gβγ subunits was unlikely to be an essential component of cell activation. However, these studies did not have adequate time resolution to define precisely the disassembly kinetics. Ternary complexes were assembled using three formyl peptide receptor constructs (wild type, FPR-Gα i2 fusion, and FPR-GFP fusion) and two isotypes of the α subunit (α i2 and α i3 ) and βγ dimer (β 1 γ 2 and β 4 γ 2 ). At saturating nucleotide levels, the disassembly of a significant fraction of ternary complexes occurred on a subsecond time frame for α i2 complexes and τ 1/2 ≤ 4 sec for α i3 complexes, time scales which are compatible with cell activation. β 1 γ 2 isotype complexes were generally more stable than β 4 γ 2 associated complexes. The comparison of the three constructs however proved that the fast step was associated with the separation of receptor and G protein and that the dissociation of the ligand or of the α and βγ subunits was slower. These results are compatible with a cell activation model involving G protein conformational changes rather than disassembly of Gαβγ heterotrimer.

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High‐Throughput Flow Cytometry

Sklar¹,

Simons²,

Waller³

et al. 2010

Pharmaceutical Sciences Encyclopedia

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This article describes the creation of a discovery team in an academic environment. The team is specifically structured to perform four integrated activities: characterizing membrane protein, formatting the materials for analysis of cell physiology and molecular interactions, performing screens of small‐molecule activity on a novel high‐throughput flow cytometric instrument platform, and integrating virtual screening with activity screening. These activities improve the overall efficiency of the discovery process.

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Detection of Epitope-Tagged Proteins in Flow Cytometry: Fluorescence Resonance Energy Transfer-Based Assays on Beads with Femtomole Resolution

Cited by 32 publications

References 19 publications

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

The development of quantum dot calibration beads and quantitative multicolor bioassays in flow cytometry and microscopy

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides

High‐Throughput Flow Cytometry

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