In vitro binding assay and in vitro competitive binding assay

The in vitro translated proteins were prepared using TNT T7 Quick Coupled Transcription/Translation System (Promega). GST-TSG101 and GST-TSGΔ154-1054 proteins were synthesized in E.coli (BL21-DE3 strain) and purified by absorption to glutathione-Sepharose (Sigma). These proteins were then incubated in buffer A (1% Triton X-100, 20 mM Tris-HCl, 140 mM NaCl, 1 mM EGTA, 10% (v/v) glycerol, 1.5 mM MgCl₂, 1 mM DTT, 1 mM sodium vanadate, 50 mM NaF, 1 μg/mL aprotinin, 10 μg/mL p-amidinophenyl methane sulphonyl fluoride hydrochloride, pH 8.0) for 2 h at 4°C. Complexes that adhered to bead-bound proteins were washed thoroughly with buffer B (20 mM Tris-HCl, 150 mM NaCl, 1% NP-40, pH 8.0), and analyzed by western blotting. For in vitro competitive binding assay, the in vitro translated HA-Tal and TSGΔ154-1054 (the competitor) were mixed with glutathione-Sepharose-immobilized GST-TSG101 in buffer A, in a total volume of 100 μL. The binding mixtures were incubated at 4°C for 2 h. Unbound proteins were removed by a subsequent washing with buffer B.