The alkyne functional group then undergoes a cycloaddition reaction with a fluorescent azide. C, S-palmitoylation levels of FLAG-MBLAC2 are measured by in-gel fluorescence (IGF) at 647 nm. Protein levels are compared by immunoblotting (IB) using a FLAG antibody. HRas, a dually palmitoylated ...
Cells were then fed overnight with 50 μm palmitic acid analog with a clickable alkyne moiety (YnPalm) or with vehicle (0.1% DMSO). Cells were lysed, and HHAT was immunoprecipitated (IP) using anti-V5 antibody. Immunoprecipitated proteins or lysates were ligated by CuAAC to AzTB. Tagged ...
The cells were first stimulated with the high dose of 0.5 μg/ml LPS together with 100 U/ml INFγ for as long as 12 h and subsequently were labeled for 8 h with alkyne-containing analogues of saturated and unsaturated fatty acids, alk-16, alk-16:1, alk-17:1, or alk-18:1, in ...
APP was pulled-down from the fractions using anti-APP C-terminal antibody (C66) prior to addition of a biorthogonal alkyne-labeled fluorescent chromophore, tetramethylrhodamine (alkyne-TAMRA; Invitrogen) via “click chemistry.” The samples were then probed with an anti-TAMRA antibody (Invitrogen)....