[18] Fluorescence methods for measuring reaction equilibria and kinetics

Dandliker, Walter B.; Dandliker, J; Levison, Stuart A.; Kelly, Robert J.; Hicks, Alexandra; White, John U.

doi:10.1016/s0076-6879(78)48020-6

Cited by 30 publications

(23 citation statements)

References 19 publications

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“…In addition, the complex is formed with antibody bound to a surface, which may cause unpredictable effects on the binding measurements. Other methods of quantifying extent of complex formation, such as surface plasmon resonance [16] or fluorescence [17], do not require a separation step but rather relate a change in the property of the antibody upon binding. These methods may require modification of the antibody prior to analysis and can also be time-consuming.…”

Section: Introductionmentioning

confidence: 99%

Measurement of antibody‐antigen dissociation constants using fast capillary electrophoresis with laser‐induced fluorescence detection

Kennedy

1997

Electrophoresis

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The dissociation constant (Kd) of a monoclonal antibody with fluorescein isothiocyanate (FITC)-labeled insulin and unlabeled insulins from several species were measured using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Kd determinations were made by separating free FITC-insulin and its complex with the antibody in equilibrated solutions in 6 s or less. The use of LIF detection allowed quantification of free and bound FITC-insulin in the picomolar range, as is required to measure Kd's below 1 nM. The Kd of FITC-insulin with the antibody was determined to be 0.25 nM by Scatchard analysis. The Kd's of the antibody with unlabeled insulins from several species were obtained by fitting bound over free FITC-insulin as a function of unlabeled insulin concentration data from a series of solutions containing a fixed concentration of FITC-insulin and antibody and variable concentrations of insulin to the expected curve derived from the equilibria and mass balance of the solutions. Kd's for the different insulins were between 0.34 and 0.64 nM.

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Section: Introductionmentioning

confidence: 99%

Measurement of antibody‐antigen dissociation constants using fast capillary electrophoresis with laser‐induced fluorescence detection

Kennedy

1997

Electrophoresis

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“…Murphy (77) has provided an excellent discussion of cytometric binding methods, and particularly non-real time analysis of endocytosis and acidification. Several general reviews on cytometric methods (75,101), polarization methods (24), and probe preparation (25) are available.…”

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confidence: 99%

Real-Time Spectroscopic Analysis of Ligand-Receptor Dynamics

Sklar¹

1987

Annu. Rev. Biophys. Biophys. Chem.

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“…The concentration of oPRL-(1-53) was determined by using absorptivity E0-1% cm = 0.46, calculated by the method of Wetlaufer (11); the concentration of oPRL-(54-199) was determined by The peptide peak (-100 mg, 2% peptide) was lyophilized and redissolved in 10 ml of 0.01 M NH4HCO3 (pH 8.4) as a stock solution. Polarization experiments were carried out with [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] Al of the stock solution plus 2 ml of the NH4HCO2 buffer. All labeling and storage procedures were carried out in darkness.…”

Section: Methodsmentioning

confidence: 99%

“…The kinetics ofoPRL fragment recombination was studied by fluorescence polarization as described (7,8). Concentrations ofFTC-oPRL-(1-53) ranged from 125 nM to 250 nM and those ofoPRL-(54-199) ranged from 62.5 nM to 125 nM.…”

Section: Methodsmentioning

confidence: 99%

“…In order to investigate this apparent dichotomy, we have examined the interaction of the fibrinolysin fragments by using fluorescence polarization (5). Although this method has most commonly been applied to antigen-antibody binding (6,7), the principles involved can be generalized to study any reaction producing a sufficiently large molecular weight change in the fluorescent species (5,7,8). The usefulness of this technique has been greatly expanded in recent years by the development of appropriate instrumentation (9) and by the characterization of fluorescein isothiocyanate as a fluorescent label (10).…”

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confidence: 99%

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Ovine prolactin: equilibrium characteristics of the recombinant molecule formed by noncovalent interaction of two fibrinolysin fragments by fluorescence polarization.

Jibson¹,

Li²,

Glaser³

1981

Proc. Natl. Acad. Sci. U.S.A.

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The equilibrium characteristics of the recombined molecule formed by noncovalent interaction of two fibrinolysin fragments of ovine prolactin (oPRL) have been studied with fluorescence polarization. Fluorescein isothiocyanate (isomer I) was used to label oPRL-(1-53), creating a fluorescent peptide indistinguishable from the unlabeled fragment inthe complementation reaction with oPRL-(54-199). The dissociation constant of the recombinant prolactin was 0.144 ,uM at 30°C, with a free energy of dissociation of 9.50 kcal/mol.

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[18] Fluorescence methods for measuring reaction equilibria and kinetics

Cited by 30 publications

References 19 publications

Measurement of antibody‐antigen dissociation constants using fast capillary electrophoresis with laser‐induced fluorescence detection

Measurement of antibody‐antigen dissociation constants using fast capillary electrophoresis with laser‐induced fluorescence detection

Real-Time Spectroscopic Analysis of Ligand-Receptor Dynamics

Ovine prolactin: equilibrium characteristics of the recombinant molecule formed by noncovalent interaction of two fibrinolysin fragments by fluorescence polarization.

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