Figure 2

In vitro and in situ PS4≡ probe condition optimization for labeling the active proteomes from Huh-7 cells. (A) Active proteomes isolated from naïve Huh-7 cells were treated at 37°C for 60 min with various concentrations of substrate based probes (PS4≡) either with or without a denaturing preheating step (Δ = 100°C, 5 min), subjected to click chemistry with RhN₃, separated by SDS/PAGE electrophoresis, and visualize by in-gel fluorescence scanning. The in vitro optimal PS4≡ concentration with the strongest signal from the active proteome and least non-specific binding from the denatured proteome was determined to be 20 μM. (B) In situ optimal labeling conditions for the naïve Huh-7 proteome to give the strong and similar banding patterns as experiments conducted in vitro was determined to be an hour incubation of 100 μM of PS4≡ probe in cell culture media. Non-specific fluorescence was observed to be negligible when either the PS4≡ probe or RhN₃ was omitted.