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Extra info for Current Protocols in Chemical Biology 2011 (Volume 3)
Because of the high background fluorescence, it is often difficult to distinguish GFP-tagged protein bound to mRNA from unbound tagged protein. In addition, the approach requires an injection of target artificial RNAs, indicating that it is impossible to visualize endogenous RNAs in living cells. From this comparison of methods, it can be concluded that the PUMbased probes are generally and widely applicable for imaging RNAs in living cells (Tyagi, 2007). Critical Parameters and Troubleshooting Validation of imaging data is important for confirming RNA localization in living cells.
This allows the user to determine the type of linkage via which fatty acid probes incorporate into cellular proteins. Sometimes, detection by streptavidin-HRP results in varying degrees of background staining, which stems from streptavidin labeling of endogenously biotinylated proteins. Therefore, care should be taken to decipher nonspecifically labeled bands on the gel, and it is critical that the researcher include negative controls, such as those lacking copper sulfate or the fatty acid probes.
B. 2002. A stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. Engl. 41:25962599. G. 2006. Proteomic identification of palmitoylated proteins. Methods 40:135142. G. 1980. Fatty acid acylation of proteins in cultured cells. J. Biol. Chem. 255:1002110024. E. F. 2004. Profiling enzyme activities in vivo using click chemistry methods. Chem. Biol. 11:535-546. , and Meldal, M. 2002. Peptidotriazoles on solid phase: [1,2,3]Triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides.