Principles and limitations of methods available for the determination of binding constants with affinity capillary electrophoresis

Busch, Markus; Kraak, J.C.; Poppe, H.

doi:10.1016/s0021-9673(97)00368-3

Cited by 111 publications

(92 citation statements)

References 38 publications

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“…The deformed protein peaks presented in electropherograms (Fig. 1) could not be attributed only to adsorption and arose due to the slow kinetics of the complexation as was also con- firmed by Busch et al (1997b) and Avila et al, (1993). Especially in the Hummel-Dreyer method, the multiple or deformed positive peaks due to protein and protein-ligand complex were not of interest because in calculations only the areas of negative peaks were considered (Sebille et al, 1978).…”

Section: Binding Measurementsmentioning

confidence: 99%

“…In electrophoretic applications of that method (Busch et al, 1997b), the capillary was filled with a buffer containing the drug. Then a small amount of sample, which contained the buffer, the drug and the protein, was injected into the capillary.…”

Section: Binding Measurementsmentioning

confidence: 99%

“…1b) a positive peak appeared which corresponded to the ligand-protein complex and to the free protein. The negative peak emerged at the migration time of the ligand and the area of that peak was directly related to the amount of ligand bound to the protein, [D b ] (Busch et al, 1997b). The amount of bound ligand was quantified from an external calibration (Sebille et al, 1978;Kraak et al, 1992).…”

Section: Binding Measurementsmentioning

confidence: 99%

“…The concentration of the free ligand, [D f ], could be set as equal to the concentration of the ligand in the buffer, (Busch et al, 1997b;Kraak et al, 1992). Hence, the following relationship holds:…”

Section: Binding Measurementsmentioning

confidence: 99%

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Binding of an Oxindole Alkaloid from Uncaria tomentosa to Amyloid Protein (Aβ1-40)

Frąckowiak

Bączek

Kaliszan

et al. 2006

Zeitschrift Für Naturforschung C

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The primary aim of this work was to determine the interactions of an oxindole alkaloid (mitraphylline) isolated from Uncaria tomentosa with -amyloid 1-40 (A 1-40 protein) applying the capillary electrophoresis (CE) method. Specifically the Hummel-Dreyer method and Scatchard analysis were performed to study the binding of oxindole alkaloids with A 1-40 protein. Prior to these studies extraction of the alkaloid of interest was carried out. Identification of the isolated alkaloid was performed by the use of thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) combined with electrospray ionization mass spectrometry (ESI-MS). The proposed approach was proved to be an efficient and accurate method for specific compound isolation and identification purposes. Moreover, analytical information from the CE approach can be considered as the valuable tool for binding constant determination. The binding constant of mitraphylline with A 1-40 protein determined by the Hummel-Dreyer method and Scatchard analysis equals K = 9.95 ¥ 10 5 m Ð1. The results obtained showed the significant binding of the tested compound with A 1-40 protein.These results are discussed and interpreted in the view of developing a strategy for identification of novel compounds of great importance in Alzheimer disease therapy.

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Section: Binding Measurementsmentioning

confidence: 99%

Section: Binding Measurementsmentioning

confidence: 99%

Section: Binding Measurementsmentioning

confidence: 99%

Section: Binding Measurementsmentioning

confidence: 99%

See 2 more Smart Citations

Binding of an Oxindole Alkaloid from Uncaria tomentosa to Amyloid Protein (Aβ1-40)

Frąckowiak

Bączek

Kaliszan

et al. 2006

Zeitschrift Für Naturforschung C

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“…Methods to determine binding constants include spectroscopy, microdialysis, calorimetry and separation techniques such as CE, etc. [13][14][15][16][17]. All methods have advantages and disadvantages.…”

Section: Introductionmentioning

confidence: 99%

Frontal analysis in microchip CE: A simple and accurate method for determination of protein–DNA dissociation constant

et al. 2007

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Equilibrium constants, such as the dissociation constant (K d ), are a key measurement of noncovalent interactions that are of importance to the proper functioning of molecules in living systems. Frontal analysis (FA) is a simple and accurate capillary electrophoresis (CE) method for the determination of K d . Microchip CE coupled with laser-induced fluorescence (LIF) detection was used to determine K d of protein-DNA interactions using the FA method. A model system of immunoglobulin E (IgE) and the IgE-binding aptamer was selected to demonstrate the capability of FA in microchip CE. Because the fluorescence emission was dependent on the dye migration velocity, the velocity of the free aptamer was adjusted to be the same as that of the aptamer-IgE complex by setting up individual separation voltage configurations for the free and bound aptamers. The ratio of the free and bound aptamers in the equilibrium mixture was directly measured from the heights of their plateaus detected at 1.0 cm from the intersection of the microchip, and no internal standard was needed. The K d of the IgE-aptamer pair was determined as 6 ± 2 nM which is consistent with the reported results (8 nM).

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Recent developments in capillary zone electrophoresis of proteins

Dolnı́k¹

1999

Electrophoresis

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Principles and limitations of methods available for the determination of binding constants with affinity capillary electrophoresis

Cited by 111 publications

References 38 publications

Binding of an Oxindole Alkaloid from Uncaria tomentosa to Amyloid Protein (Aβ1-40)

Binding of an Oxindole Alkaloid from Uncaria tomentosa to Amyloid Protein (Aβ1-40)

Frontal analysis in microchip CE: A simple and accurate method for determination of protein–DNA dissociation constant

Recent developments in capillary zone electrophoresis of proteins

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