Ligand-Probe Conjugate Crosslinking Protocol

Cells were washed with cold PBS three times, and incubated with ligand-probe conjugate solution for 10 min at 4 °C. The solution was then removed and replaced with PBS. For photocrosslinking, the cells were UV irradiated in the UVP CL-1000L UV Crosslinker (365 nm) for 5 min at 4 °C. For click chemistry crosslinking, the catalyst buffer was prepared according to previous reports with slight modifications^{18 (link),19 (link)}. Briefly, CuSO₄, THPTA, aminoguanidine and a freshly prepared solution of sodium ascorbate were sequentially mixed and added to PBS to final concentrations of 50 μM, 250 μM, 1 mM, and 2.5 mM, respectively. This catalyst buffer was placed on ice for 10 min and then added to cells for 15 min at 4 °C.