Streptavidin Bead Elution and In-Gel Tryptic Digestion

For the APEX2 experiments, streptavidin beads were eluted by boiling with 2× Laemmli loading buffer (Biorad, 161 to 0737) containing 20 mM DTT and 2 mM biotin. The eluate was then run on a 4% to 15% Tris-glycine gel (Biorad, #4561084) for 30 min at 50 V. Gel slices were then fixed according to Mackinder et al. (2017) (link). In-gel tryptic digestion was performed after reduction with 10 mM dithioerythritol and 50 mM S-carbamidomethylation with iodoacetamide. Gel pieces were washed 2 times with aqueous 50% (v/v) acetonitrile containing 25 mM ammonium bicarbonate and then once with acetonitrile and dried in a vacuum concentrator for 20 min. A 500-ng aliquot of sequencing-grade trypsin (Promega) was added prior to incubation at 37 °C for 16 h.