Streptavidin dylight 800

1.5 × 10⁸ epimastigotes were pelleted at 2060 x g for 10 minutes, resuspended in 1 ml of LIT and incubated with 50 µM biotin for 10 minutes at 37^oC. Parasites were washed twice with ice-cold PBS then resuspended in 1 ml cell lysis buffer ^{55 (link)} (0.5% Nonidet P-40, 500 mM NaCl, 5 mM EDTA, 1 mM DTT, 50 mM Tris-Base, 0.4% SDS, pH 7.4) with Roche cOmplete^™ Protease Inhibitor (Sigma-Aldrich, St. Louis, Missouri, USA), 100 μM PMSF and 10 μM TLCK. Lysates were sonicated using 3 pulses of 30 seconds at 100% amplitude (Q700 sonicator, QSONICA, Newton, Connecticut), with 15 sec breaks between to cool the tubes on ice. Samples were centrifuged at 16,000 x g for 20 minutes at 4°C and the supernatant was collected. Aliquots of clarified lysate (3 × 10⁶ parasite equivalents) were resolved by SDS-PAGE (Mini-PROTEAN®TGX protein gel; Bio-Rad, Hercules, California, USA), transferred to PVDF membrane (Immobilon^®-FL, MilliporeSigma, Burlington, Massachusetts, USA) and probed with Streptavidin DyLight^™ 800 (Thermo Fisher Scientific, Waltham, Massachusetts, USA) to detect biotinylated proteins.