Unlabeled ligand

Lazareno, S., and Birdsall, N. J. M. (1995). Detection, quantitation, and verification of allosteric interactions of agents with labeled and unlabeled ligands at G protein-coupled receptors Interactions of strychnine and acetylcholine at muscarinic receptors. Mol. Pharmacol. 48 362-378. 

More recent applications comprise, for example, the identification of the binding site of 18 kDa human cardiac troponin C for the drug bepridil [36]. For this study, the unlabeled ligands were bound to selectively [13CH3-Met, Phe-d8]-labeled protein (Fig. 17.8). First, the 13CH3-Met signals of troponin C were easily identified from 13C-HSQC spectra. In a 2D NOESY spectrum with 13C-editing in one dimension, intermolecular NOEs could... 

The appropriate definition of nonspecific binding is essential prior to characterization of the kinetic and equilibrium properties of the binding interaction. As a rule, nonspecific binding can be defined using a concentration of the unlabelled ligand that is 100 times its Ka value for the sites of interest. Failure to appropriately define nonspecific binding will invalidate the determination of the binding parameters. 

After the interaction between the ligand and receptor has reached equilibrium, dissociation kinetics are initiated either by dilution or by the introduction of unlabelled ligand which competes for the receptor binding sites. The purpose, in both cases, is to ensure that once the radiolabelled ligand has dissociated from the receptor, it is not able to reassociate with the receptor. This allows the following reaction to occur in isolation ... 

Sklar, L. A., Sayre, J., McNeil, V. M., and Finney, D. A. (1985). Competitive binding kinetics in ligand-receptor-competitor systems. Rate parameters for unlabeled ligands for the formyl peptide receptor. Mol. Pharmacol. 28, 323-330. 

Dalvit C, Cottens S, Ramage P, Hommel U, Half-filter experiments for assignment, structure determination and hydration analysis of unlabelled ligands bound to 13C/15N labelled proteins, J. Biomol. NMR, 13 43-50, 1999. 

Fig. 6.2. Kinetic competition labeling for optimal discrimination of mutant clones. Both wild-type and mutant clones are labeled to saturation, and at time zero excess unlabeled ligand competitor is added. Single cell fluorescence drops exponentially to background levels, which are the same for both mutant and wild-type cells. Optimal discrimination is obtained at an intermediate competition time.

Another possibility is to use half-edited/ half-filtered 2D experiments to detect NOEs that specifically involve interactions between protons attached to or and those that are not. This approach is used, for example, to detect intermolecular NOEs between a labeled protein and an unlabeled ligand. Examples of isotope editing/filtering are given in section 3.2.4. 

Modulation of the activity response by competing, unlabeled immu-noreactants may be achieved by two approaches (i) the labeled and unlabeled ligand compete simultaneously for the available binding sites of the antibodies (equilibrium techniques) and, (ii) the unlabeled ligand (test or calibration sample) is reacted first with the available binding sites and the number of occupied binding sites is estimated by the subsequent addition of the labeled reactant (sequential saturation). These methods are conceptually different and... 

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