By Bruce G. Allen, Terence E. Hébert
Nuclear G-Protein Coupled Receptors: tools and Protocols is a compilation of a few conceptual and methodological points vital for the validation and characterization of intacrine signaling structures. so far, the best-characterized intracrine signaling method is that of angiotensin II (Ang II), lined intensive in quite a few chapters. technique to check the subcellular localization and serve as of GPCRs and different signaling structures is supplied, in addition to a number of chapters concentrating on tools designed to appreciate signaling mediated via nuclear and different inner GPCRs. tools also are defined to review the formation of moment messengers similar to cAMP and to check the trafficking of receptors from the mobilephone floor. Written within the profitable Methods in Molecular Biology sequence layout, chapters comprise introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, without difficulty reproducible protocols, and notes on troubleshooting and averting recognized pitfalls.
Authoritative and simply available, Nuclear G-Protein Coupled Receptors: equipment and Protocols seeks to serve either pros and newbies with cutting-edge methods to signify what's changing into a typical topic in mobile signaling.
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Extra resources for Nuclear G-Protein Coupled Receptors: Methods and Protocols
H-Gly-OH. 20 David Chatenet et al. 6. N-α-Fmoc-L-aspartic acid-α-tert-butyl ester. 7. N-α-Fmoc-L-glutamic acid-α-tert-butyl ester. 8. N-α-Fmoc-L-cysteine-OH. 9. N-α-Boc-L-aspartic acid-α-tert-butyl ester. 10. N-α-Boc-L-glutamic acid-α-tert-butyl ester. 11. O - ( 6 - c h l o r o b e n z o t r i a z o l - 1 - y l ) - N , N , N ′ , N ′ tetramethyluronium hexafluorophosphate. 12. N-hydroxybenzotriazole. 13. Wang resin. 14. Rink amide resin. 15. 2-Chlorotrityl chloride resin. 4. Reagent A: Dissolve 1 g of ninhydrin in 20 mL of ethanol.
As a final step, an Fmoc-protecting group is added on the free amino terminus of the amino acid derivatives. 5. ) in H2O-acetone (1:1) over a period of 1 h while the pH is kept between 9 and 10 by the addition of 1 M Na2CO3 solution. Stirring is continued overnight and progression of the reaction is monitored by RP-HPLC. 6. Upon completion, EtOAc is added and the mixture is acidified with 6 M HCl. The organic layer is washed with H2O, dried over anhydrous MgSO4, and concentrated in vacuo. If necessary, the resulting residue is purified by flash chromatography (SiO2).
Bead loading and microinjection have certain limitations. Bead loading needs high concentrations of caged compounds and can only be employed on small numbers of cells. Microinjection permits the loading of only one cell at a time . Moreover, both methods require the co-loading of inert fluorescent molecules such as fluorescein-dextran or rhodamine-dextran to evaluate the success of loading. Here, we described the use of cell-permeant caged ligands to activate or block the intracellular pool of receptors while sparing the cell surface receptors.