Streptavidin irdye680lt

Growth, APEX2-mediated labeling, and enrichment of biotinylated proteins was performed as reported^{23 (link)}. Briefly, Mtb/APEX2m and Mtb/Sec-APEX2m were cultured in liquid medium to an optical density at 600 nm (OD₆₀₀) of 0.8-1. Mtb was then subclutured to OD₆₀₀ 0.25 in 50 mL (Mtb/APEX2m) or 250 mL (Mtb/Sec-APEX2m) with or without 2 mM theophylline and cultured for a further 72 h. Cells were harvested by centrifugation at 2500 xg for 10 min, resuspended at 10X concentration in 5 mL (Mtb/APEX2m) or 25 mL (Mtb/Sec-APEX2m) fresh medium containing 1 mM biotin-phenol (Iris Biotech), and incubated at 37 °C for 30 min. After harvesting by centrifugation, cells were resuspended in PBS and lysed by bead beating. Clarified lysates were then extracted with 1 % w/v dodecyl-maltoside at 4 °C for 1 h. Pre-washed neutravidin beads (Pierce PI29201) in PBS were added to lysates, incubated with gentle mixing at 22 °C for 1 h, and then washed with PBS. Enrichment and yield were confirmed by analyzing a fraction of the beads by SDS-PAGE followed by streptavidin Western blot (Streptavidin IR-Dye 680 LT; LI-COR Odyssey detection) and silver stain (Pierce), respectively.