By Bruce G. Allen, Terence E. Hébert
Nuclear G-Protein Coupled Receptors: tools and Protocols is a compilation of a couple of conceptual and methodological elements vital for the validation and characterization of intacrine signaling platforms. thus far, the best-characterized intracrine signaling approach is that of angiotensin II (Ang II), coated extensive in numerous chapters. technique to check the subcellular localization and serve as of GPCRs and different signaling structures is equipped, in addition to various chapters targeting tools designed to appreciate signaling mediated via nuclear and different inner GPCRs. equipment also are defined to check the formation of moment messengers resembling cAMP and to check the trafficking of receptors from the cellphone floor. Written within the winning Methods in Molecular Biology sequence layout, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, with no trouble reproducible protocols, and notes on troubleshooting and heading off recognized pitfalls.
Authoritative and simply obtainable, Nuclear G-Protein Coupled Receptors: tools and Protocols seeks to serve either pros and newcomers with state of the art ways to signify what's changing into a typical subject matter in mobile signaling.
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Additional info for Nuclear G-Protein Coupled Receptors: Methods and Protocols
Hence, uncaging 40 Clémence Merlen et al. of the entire cell would affect the kinetics of the experiment as well as expose the cell to high levels of UV. To minimize UV damage, as well as provide a more rapid and spatially controlled release of ligand, the microscope is configured to scan a 60 μm2 rectangular area adjacent to or overlapping the nucleus. The position of the nuclei (most ACVMs are binucleated) is revealed by the DRAQ5 fluorescence. 7. By placing the uncaging rectangle (see Note 6) at various distances from the nucleus, while quantifying Fluo-4 emissions within the nucleus, one may ascertain the ability of the uncaged ligand to diffuse to the nucleus.
06 % TFA, v/v). 2. Solvent B: Acetonitrile (ACN). 3. 6 mm) column (Phenomenex, Torrance, CA). 4. 2 mm) column (Phenomenex). 3 Methods To prevent unwanted photocleavage, all reactions should be carried out in the dark by putting aluminum foil around the reaction vessel. The general procedure described herein employs conditions that minimize racemization of the amino acid and provides a simple, high-yield route to caged amino acids. 1 Preparation of Caged Fmoc-Tyr (4-ODMNB)-OH ( See Note 1)  1.
2. Light-Activated Probes for Cellular Signaling 29 3. The Fmoc-protected amino acid next to the N-α-caged serine is coupled to the β-hydroxyl group of serine using 1 equiv. of N,N′-diisopropylcarbodiimide (DIPCDI) and DMAP in DCM for 2 h. This reaction is repeated at least four times to ensure completion. 4. The peptide synthesis is then resumed as described above and after cleavage from the resin, the peptide is purified by RP-HPLC. 4 Notes 1. This synthetic route can also be applied to prepare FmocSer(DMNB)-OH and Fmoc-Thr(DMNB)-OH.