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Related Concept Videos

The Equilibrium Binding Constant and Binding Strength02:18

The Equilibrium Binding Constant and Binding Strength

The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:
The Equilibrium Binding Constant and Binding Strength02:18

The Equilibrium Binding Constant and Binding Strength

The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:
Ligand Binding Sites02:40

Ligand Binding Sites

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
Ligand Binding Sites02:40

Ligand Binding Sites

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the...
Ligand Binding and Linkage00:49

Ligand Binding and Linkage

Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...
Ligand Binding and Linkage00:49

Ligand Binding and Linkage

Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...

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An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions
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An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions

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Ligand binding assays at equilibrium: validation and interpretation.

Edward C Hulme1, Mike A Trevethick

  • 1Division of Physical Biochemistry, MRC National Institute for Medical Research, Mill Hill, London, UK. ehulme@nimr.mrc.ac.uk

British Journal of Pharmacology
|February 6, 2010
PubMed
Summary
This summary is machine-generated.

This review details factors impacting data interpretation in ligand binding assays, focusing on determining equilibrium dissociation (K(d)) and inhibitor (K(dA)) constants. It provides strategies to avoid errors and ensure reliable affinity measurements for drug interactions.

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Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction
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Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction

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Area of Science:

  • Biochemistry
  • Pharmacology
  • Analytical Chemistry

Background:

  • Ligand binding assays are crucial for characterizing receptor-ligand interactions.
  • Accurate determination of equilibrium dissociation (K(d)) and inhibitor (K(dA)) constants is essential for drug discovery and development.
  • Experimental design and assay conditions significantly influence data reliability.

Purpose of the Study:

  • To review factors affecting data interpretation in ligand binding assays under equilibrium conditions.
  • To provide strategies for accurate determination of K(d) and K(dA) values.
  • To discuss overlooked factors influencing affinity measurements.

Main Methods:

  • Development of the basic theory for receptor ligand-radiotracer interactions.
  • Discussion of experimental design to avoid ligand depletion and ensure equilibrium attainment.
  • Exploration of strategies to correct errors in K(d) and K(dA) calculations.

Main Results:

  • Inappropriate experimental design can lead to ligand depletion and non-attainment of equilibrium, distorting K(d) and K(dA) values.
  • Adequate specific binding signal is necessary for accurate affinity constant measurement, imposing limits on measurable affinities.
  • Assay methodology can distinguish between competitive and allosteric ligand-receptor interactions.

Conclusions:

  • Reliable determination of K(d) and K(dA) requires careful experimental design and adherence to equilibrium conditions.
  • Understanding assay limitations, such as the specific binding signal range, is critical.
  • Buffer composition and temperature are often overlooked factors that significantly impact affinity measurements and drug interaction understanding.