By Michelle Qume (auth.), Mary Keen (eds.)
In Receptor Binding strategies, certain experimental protocols devised by way of specialist practitioners display the ability of radioligand binding for learning a large choice of receptors. The tools should be utilized to G protein-coupled receptors, receptors with fundamental ion channels, and websites except receptors, akin to guanine nucleotide binding proteins, enzymes, transporters, and mRNA. the person protocols surround many alternative arrangements, starting from solubilized receptors, via membrane arrangements, intact cells, and autoradiography in tissue slices, to puppy and SPECT research of binding in dwelling human mind. besides the distinctive, simply reproducible directions, the protocols additionally take into consideration the issues inherent within the simple binding strategy, resembling universal experimental artifacts and the definition of receptor websites, in addition to difficulties linked to using specific ligands and receptor preparations.
Receptor Binding strategies indicates sincerely how those thoughts may be tailored to many alternative functions and completely used in drug discovery courses. it is going to be of excessive price not just to researchers new to the sphere of receptor binding, but additionally to more matured employees, serving as an authoritative bench guide replete with optimalized useful equipment and protocols.
Pharmacologists, neuroscientists, mobilephone biologists, molecular biologists, and biochemists
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Sample text
Saturation Binding Studies to Determine Receptor Density (Bma~n (see Note 2) 1. Prepare the tritiated ligands at 10X the final concentration required in the assay tube so that 50 ktL is added in a final assay volume of 500 ~tL. For the concentrations and assay layout, see Table 2. 2. A range of concentrations of [3H]2-BFI, [3H]idazoxan, and [3H]-p-aminoclonidine are added in a volume of 50 ~L. Make up the latter two ligands in the presence of 50 ktM rauwolscine to preclude binding to Otz-adrenoceptors, yielding a final concentration of 5 ktM rauwolscine in the assay tube.
Centrifuging the supernatant (S1, the crude membrane fraction, enriched in myelin, synaptosomes, and mitochondria) produces the P2 pellet and the Se supernatant. Following hypotonic shock (to lyse nerve endings) and further centrifugation of the P2 pellet, the pellet becomes a bilayer. The brownish, lower layer of the bilayer is the mitochondrial and myelin fraction, whereas the combined supernatant and upper layer are the crude synaptic fraction rich in synaptic membranes. This crude synaptic fraction then undergoes further resuspension and centrifugation to wash the membranes, reducing endogenous ligand content, which enhances binding.
Resuspend the pellet in 25 mL binding buffer and incubate at room temperature for 45 min before centrifuging for 10 min at 9000g at 4~ 6. Discard the supernatant. 7. 2 mg/mL. One whole rat brain should yield enough tissue for about 40 tubes (34 mL). 8. Use the membranes immediately for the radioligand binding assay, but save a small amount of each preparation for later determination of protein content ( s e e Note 11). 3. Radioligand Binding Assay 1. Each assay condition is determined in triplicate.