S9390

Cold APC purification buffer [100 mM NaPi, Sigma #S3139 and Sigma #S9390, 300 mM KCl, Sigma #P9333, 5 mM MgCl₂ × 6 H₂O, Sigma #M2670, 0.001% Brij35 Thermo Fisher Scientific, #28316, 2.5 mM dithiothreitol (DTT), Sigma #D0632, 2.5 mM EDTA, Sigma #EDS] supplemented with protease inhibitors (Roche, #5056489001) and deoxyribonuclease I (DNaseI) (Roche, #10104159001) was added to frozen SF21 cell pellets expressing an APC construct. The pellet was thawed in a room temperature water bath and resuspended. The lysate was clarified by centrifugation (184.000g, 45 min, 4°C), and the supernatant was applied to a 5-ml HiTrap immunoglobulin G Sepharose FF column (GE Healthcare, #28-9083-66). After washing with APC purification buffer, GST (glutathione S-transferase)–TEV protease was added to the column, and the column was left at 4°C overnight to cleave APC off its column-bound affinity and solubility tags. The next day, APC was eluted with APC purification buffer and labeled with SNAP-reactive dye (NEB #S9105S). After SNAP labeling, APC was concentrated using Vivaspin concentrators (Sartorius), ultracentrifuged (280,000g, 10 min, 4°C), and gel filtered using a Superose6 10/300 GL column (GE Healthcare). Peak fractions were pooled, and the labeling ratio was determined using NanoDrop (Thermo Fisher Scientific). The protein was aliquoted on ice and snap frozen in liquid nitrogen.

Cold APC purification buffer (100 mM NaPi, Sigma #S3139 and Sigma #S9390, 300 mM KCl, Sigma #P9333, 5 mM MgCl₂ × 6 H₂O, Sigma #M2670, 0.001% Brij35 Thermo Fisher Scientific #28316, 2.5 mM DTT, Sigma #D0632, and 2.5 mM EDTA, Sigma #EDS) supplemented with protease inhibitors (Roche, #5056489001) and DNaseI (Roche, #10104159001) was added to frozen E. coli cell pellets expressing an APC-ARM construct. The pellet was thawed and resuspended. The lysate was clarified by centrifugation (184,000g, 45 min, 4°C), and the supernatant was applied to a StrepTrap column (GE Healthcare, 28-9075-48). The column was washed with APC purification buffer, and the protein was eluted in APC purification buffer supplemented with 10 mM d-desthiobiotin (Sigma, D1411-1G). Next day, the protein was incubated with 3C protease (Speed Biosystems, YCP1208) for 1 hour at 16°C. After SNAP labeling, the protein was concentrated and ultracentrifuged as described above and gel filtered using a GE Superdex200 10/300 column. Peak fractions were pooled, and the labeling ratio was determined using NanoDrop. The protein was aliquoted on ice and snap frozen in liquid nitrogen.