| Assay Method Information | |
| | Competitive ABPP Assays in Proteomes with FP-alkyne |
| Description: | For in vitro experiments, proteomes were diluted to 1 mg/mL in PBS (pH 7.5, 50 μL total reaction volume), doped with 1 μM recombinant PAFAH1b2 and PAFAH1b3, and incubated with compound at the indicated concentrations (1 μL of a 50x stock in DMSO) for 30 min at 37 °C, followed by labeling with 1 μM FP-alkyne (1 μL of a 50x stock in DMSO) for 10 min at 25 °C. Total protein concentrations for each fraction were adjusted to 1 mg/mL in PBS (50 μL total reaction volume). Samples were then subjected to click chemistry by the addition of 1 mM CuSO4 (1 μL of a 50x stock in DMSO), followed by TBTA (3 μL of a 17x stock in 4:1 t-butanol:DMSO), then 25 μM Rh-N3 (1 μL of a 50x stock in DMSO) and 1 mM TCEP (1 μL of a 50x stock in DMSO). Reactions were vortexed, incubated for 1 h at 25 °C, and quenched with 4x SDS-PAGE loading buffer and analyzed by in-gel fluorescence scanning. |
| Affinity data for this assay | |
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