Ultra-Rapid Fluorescent Labelling of Proteins

Rinderknecht, H.

doi:10.1038/193167b0

Cited by 337 publications

(76 citation statements)

References 2 publications

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“…A fluorescent label can be attached using methods that are wellestablished in protein chemistry. 17 The parent PEG samples with weight-average molecular weights 2200, 5000, 10 800, 20 100, and 30 500 g mol -1 (degree of polymerization N ) 48, 113, 244, 456, and 693, respectively) and ratio of weightaverage to number-average molecular weight Mw/Mn ) 1.01-1.03 were purchased from Shearwater Polymers, Inc. The molecules contained the methoxy group at one terminus and amino group at the other one (PEG-NH2).…”

Section: Methodsmentioning

confidence: 99%

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Surface Diffusion of Poly(ethylene glycol)

et al. 2002

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We report direct measurement of the center-of-mass diffusion coefficient, D, of uncharged flexible linear chains adsorbed at the solid-liquid interface at dilute surface coverage. We find D ∼ N -3/2 (N is degree of polymerization) when N was varied by more than an order of magnitude (N ) 48, 113, 244, 456, and 693) and the scatter of the data was low. The experimental system was poly(ethylene glycol), PEG, adsorbed from dilute aqueous solution onto a self-assembled hydrophobic monolayer, condensed octadecyltriethoxysilane. The method of measurement was fluorescence correlation spectroscopy of a rhodamine green derivative dye that was end-attached to one sole end of the adsorbed PEG chains. The observed scaling implies the diffusion time τ ∼ N 3 if Rg ∼ N 3/4 as expected for a chain in good solvent in two dimensions (Rg is the radius of gyration), but a variety of other theoretical approaches to describe the dynamical scaling are also plausible. The multiplicity of plausible dynamical transport scenarios is compounded by the fact that polymer diffusion is sensitive to chain conformation on the surface which is not directly observable. Various theoretical scenarios are explored, and the need for new experiments, theory, and computer simulation studies to allow definitive interpretation of this observation of simple and clean fractional power law scaling is emphasized.

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Section: Methodsmentioning

confidence: 99%

“…The procedure was based on the well-known protocol for labeling of proteins, which involves chemical reaction between the isothiocyanate group of the label and amino group of the protein. 17,18 In this work the labeling was done in organic solvent.…”

Section: Methodsmentioning

confidence: 99%

Surface Diffusion of Poly(ethylene glycol)

et al. 2002

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“…37 -38 In the present study, we investigated the possibility that thrombin and/or other platelet activators change the platelet surface expression of GPIV in vitro and in vivo. 48 except for FITC-conjugated OKM5, which was provided by Dr Rao (Ortho). The biotinylated and FTTC-conjugated antibodies functioned normally, in that their binding to platelets was saturable at antibody concentrations similar to that of unlabeled antibodies.…”

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confidence: 99%

Platelet activation results in a redistribution of glycoprotein IV (CD36).

Michelson

Wencel-Drake

Kestin

et al. 1994

Arterioscler Thromb

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To investigate the possibility that thrombin and/or other platelet activators change the platelet surface expression of glycoprotein IV (GPIV, CD36), we used a panel of five GPIV-specific monoclonal antibodies (OKM5, 5F1, FA6-152, 8A6, and F13) directed against different epitopes. All these antibodies bound to resting platelets in a concentration-dependent and saturable manner, as determined by flow cytometry of washed platelets. Thrombin (1 U/mL) induced an approximately twofold increase in the platelet surface binding of each of these monoclonal antibodies. Immunofluorescence microscopy demonstrated an internal pool of GPIV that, after thrombin stimulation, redistributed to the platelet surface. In a whole-blood fiow-cytometric assay, a-thrombin and the G lycoprotein (GP) IV, also known as GPIIIb and CD36, is present on the surface of human platelets and other cells, including endothelial cells, monocytes, erythrocytes, and megakaryocytes. 1 GPIV is composed of a single highly glycosylated polypeptide chain with an apparent molecular mass of 88 kD.2 - 3 The structure of GPIV has several homologies with a recently described lysosomal integral membrane protein (LIMP II), suggesting that GPIV and LIMP II belong to a new gene family. 4 There are between 12 000 and 25 000 copies of GPIV on the platelet surface. - 6GPIV is very resistant to protease digestion. 78 GPIV may play a number of important roles in platelet function and pathophysiology. First, GPIV has been reported to be a collagen receptor involved in the initial phase of platelet-collagen adhesion. 910 Second, GPIV has been reported to be a receptor for thrombospondin 3 ' 11 and may therefore have a role in thrombospondin-dependent platelet aggregation 1213 and thrombospondin-dependent platelet-monocyte adhesion. 14 Third, platelet GPIV is physically associated with the fyn, lyn, and yes protein tyrosine kinases and may be involved in platelet signal transduction.1 -15 Fourth, GPIV is a receptor for the adhesion of Plasmodium falciparumAniected erythrocytes. 1617 Fifth, GPIV may have a role in the pathophysiology of thrombotic thrombocytopenic purpura (TTP), because the plasma of a Received November 5, 1993; revision accepted April 26, 1994 The platelet-specific antigen Nak* is present on GPIV. 23- 24 The Nak a -negative phenotype occurs with a high incidence (=3%) in Japanese blood donors 25 but occurs in only 0.2% of US blood donors.1 Because they lack detectable GPIV, 2324 the platelets of Nak'-negative individuals have been used to study the role of GPIV in platelet function. Nak'-negative individuals show reduced adhesion to collagen in flowing whole blood. 10The lack of evidence of a hemostatic defect in Nak'-negative individuals suggests that binding of collagen to other proteins (eg, the GPIa-IIa complex 26 and GPVI 27 ) may be sufficient to avoid an overt bleeding diathesis. Thrombospondin binding to the platelets of Nak'-negative individuals appears to be normal, 2829 which calls into question the role of GPIV as a thrombospondin...

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“…50 mg of enzyme and 10 mg FITC were mixed with 2 mL pH 8.5 carbonate -bicarbonate buffer. After shaking 3 min the mixture was centrifuged at 5000 g and the supernatant passed through a Sephadex G25 column [6]. The fraction containing the fluorescent enzyme was freeze-dried.…”

Section: Catalyst Preparation By Labeling With Fluorescein Isothiociamentioning

confidence: 99%

Spectroscopic methods of studying enzyme action in wool fibre

Fogorasi

Heine

2006

Open Chemistry

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Enzymes may be used to develop an environmentally friendly alternative to the conventional polluting technologies in textile finishing. The action of the unlabeled and fluorescent labeled proteolytic enzymes subtilisin and trypsin on wool was examined. Scanning electron micrographs and a diffusion study, based on fluorescence microscopy, localized the enzymatic attack on the fiber. A kinetic study was carried out by monitoring the amino acid content of the treatment liquor. Enzymatic action is not confined to the fiber surface. To limit attack to the surface and reduce wool damage new treatment methods such as enzyme immobilization onto a solid carrier must be investigated.

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Ultra-Rapid Fluorescent Labelling of Proteins

Cited by 337 publications

References 2 publications

Surface Diffusion of Poly(ethylene glycol)

Surface Diffusion of Poly(ethylene glycol)

Platelet activation results in a redistribution of glycoprotein IV (CD36).

Spectroscopic methods of studying enzyme action in wool fibre

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