Tris 1 benzyl 1h 1 2 3 triazol 4 yl methyl amine

Aminoguanidine hydrochloride, copper(II) sulfate (CuSO₄), (+)-sodium l-ascorbate, tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, THPTA, l-glutamate, and chemicals used for synthesis were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless stated otherwise. LY379268, LY354740, DCG-IV, L-CCG-I, and BINA were purchased from Tocris Bioscience (Bristol, UK). O⁶-benzylguanine and amine derivatives of Lumi4-Tb, green (fluorescein derivative), red (DY647 derivative) fluorophores, as well as Tag-lite buffer were from PerkinElmer (Codolet, France). 4-PrF was initially synthesized according to (31 (link)) and later purchased from Iris Biotech GmbH (Marktredwitz, Germany). BTTAA was synthesized as described in (40 (link)). pAz-Lumi4-Tb, pAz-green, and pAz-red were synthesized as described in the Supplementary Protocol. pAz-AF488, pAz-AF546, and pAz-AF647 were purchased from Jena Bioscience (Jena, Germany). LMNG and CHS tris salt were purchased from Anatrace (through CliniSciences, France). GDN was purchased from Avanti Polar Lipids through Merck.

HEK293T cells were transfected with myc-tagged WT or SAAX mutant small GTPases. Ninety minutes following transfection, cells were metabolically labeled with 10 μM C15AlkOPP isoprenoid probe for 18 h. Cells were then harvested and lysed as described (58 (link)). Briefly, cell pellets were suspended and lysed by sonication in PBS + 1% SDS. Protein concentrations were determined using bicinchoninic acid Assay (Thermo Fisher Scientific, 23225), following the manufacturer’s protocol. Proteins (100 μg/100 μl) were subjected to click reaction with 25 μM TAMRA PEG₃-N₃ (BroadPharm), 1 mM Tris(2-carboxyethyl)phosphine hydrochloride (Sigma-Aldrich), 0.1 mM Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (Sigma-Aldrich), and 1 mM CuSO₄ in the dark with gentle shaking for 1 h at room temperature. Proteins were precipitated using ProteoExtract Protein Precipitation Kit (Calbiochem, cat. no. 539180), following the manufacturer’s protocol. Resulting protein pellets were suspended in Laemmli sample buffer, boiled for 5 min, run on 12% SDS-PAGE gels, and transferred to PVDF. Fluorescence and immunoblotting images were obtained using Azure c600 and analyzed using AzureSpot software (Azure Biosystems). Fluorescence optical densities of overexpressed proteins were normalized to the optical density of myc-tagged protein.