Cold ligands

Cold ligand for competition binding assay Increasing concentration from a 1 mg/mL stock solution (see Note 6). 

Cold ligand for saturation binding assay 100-500 x the concentration of the radiolabeled chemokine. 

The gel to be coupled is suspended uniformly, transferred to a sintered glass funnel and washed with 3 volumes of iso-propanol and 3 volumes of cold distilled water (less than 20 min). The cold ligand solution (0.5-1 volume) in 100 mM NaHCOsis mixed gently with the gel for 1 h at room temperature. This is followed by an incubation (1 h) with ethanolamine-HCl buffer, pH 8.0. The gel is washed and ready for use. Ligands not soluble in water may be used in methanol, ethanol, acetone or DMF. 

Experimental determination of and Bmax for a given radioligand and receptor preparation requires incubation of various concentrations of the radioligand with a fixed concentration of receptors this is the so-called saturation experiment. Such experiments can be performed in the absence (control) or presence of various concentrations of non-radioactive ( cold ) ligand or drug — also termed... 

Figure 8.8 Dissociation of ( H]iloprost from the receptor of NCB-20 membranes. An equilibrium was established at 20°C for 20 min with 15 nmol [ HJiloprost. At time zero, 4fimo L cold ligand was added, and the decrease in specific binding measured (A). A semilogarithmic plot of the data is shown in B. (Reproduced from reference 28 with permission)...

Table 29.1.4 Comparison between electro-physiological and [ H]-7 displacement potencies for different neonicotinoids 6-9, 13 and ( )-epibatidin (16) on insect nAChRs. Electrophysiological data [ECso and relative (agonist) efficacy] were obtained from neuron cell bodies isolated from the CNS of H. virescens. EC50 and relative efficacy values represent the mean of separate experiments on different neurons. Inhibition of [ H]-7 binding to nAChR in housefly head membrane preparations by the compounds is expressed as piso (piso values (= -log M) correspond to the concentration of cold ligand displacing 50% of bound [ H]-7 from housefly head membranes).

Increasing concentrations of cold ligand in HEPES binding buffer supplemented with 0.5% BSA (HBB/BSA), 25 pl/well, is added to a... 

Figure 2 Time course of radioligand binding kinetics. Radioligand is introduced to the system (A) and allowed to bind (B) until equilibrium is reached (Q. Free radioligand is removed or outcompeted by an excess of orthosteric cold ligand (D) which results in a time-dependent dissociation of radioligand (E).

Figure 6 Displacement binding. Radioligand and cold ligand compete for receptor binding. A single radioligand concentration is displaced by increasing concentrations of competitive ligand.

Ligands Both radiolabeled and cold ligands should be stored at -20°C in the dark in DMSO or ethanol. [ H]All-tra 5-retinoic acid is commercially available from NEN [ H]Am80 was prepared at Amersham (9). 

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