Peroxidase conjugated anti igg

Lysates were prepared in CHAPS buffer (10 mM Tris-HCl [pH 7.4], MgCl₂ 1 mM, EGTA 1 mM, CHAPS 0.5%, glycerol 10%, β-mercaptoethanol 5 mM, PMSF 1 mM) containing protease inhibitor cocktail. Cell lysates and exosomes were subjected to electrophoresis on SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Protran Whatman, Dassel, Germany). After blocking in 5% dry milk in PBS1X, membranes were hybridised with primary antibodies: goat anti-mouse IgG HRP (Amersham Biosciences, Milan, Italy), anti-Tsg101(4A10, GeneTex, Irvine, CA, USA) and anti-GAPDH (GA1R, Santa Cruz Biotechnology, Dallas, TX, USA) monoclonal antibodies. After incubation with appropriate peroxidase-conjugated anti-IgG (Amersham Biosciences, Milan, Italy), membranes were revealed using the ECL Chemiluminescent Substrate (ThermoFisher Scientific, Waltham, MA, USA).

To perform Western blot analysis, the plasmatic exosomes pellet was resuspended in 1 ml of PBS and it was further purified by using 30% sucrose in deuterium oxide (D₂O, ACROS Organics, fisher scientific, USA) density gradient ultracentrifugation for 18 h at 110,000 g, in order to eliminate contaminants⁴⁶ (link)^,47 . Density gradient ultracentrifugation was performed by using TH-641 Rotor (ThermoFisher Scientific, USA). The 12 fractions obtained were washed in PBS for 1 h at 110,000 g and then were resuspended in CHAPS buffer 1x for subsequent experimental analysis.
Lysates were prepared in CHAPS buffer (10 mM Tris-HCl [pH 7.4], MgCl₂ 1 mM, EGTA 1 mM, CHAPS 0.5%, glycerol 10%, β-mercaptoethanol 5 mM, PMSF 1 mM) containing protease inhibitor cocktail. Exosomes lysates were subjected to electrophoresis on SDS polyacrylamide gels and transferred to nitrocellulose membranes (ProtranWhatman, Dassel, Germany). After blocking in 5% dry milk in PBS 1X, membranes were hybridised with primary antibodies: M75⁴⁸ (link), anti-CD81 (B-11, Santa Cruz Biotechnology, USA), anti-Alix (3A9, ThermoFisher Scientific, Waltham, MA, USA) mouse monoclonal antibodies. After incubation with appropriate peroxidase-conjugated anti-IgG (AmershamBiosciences, Milan, Italy), membranes were revealed using the ECL Chemiluminescent Substrate, (ThermoFisher Scientific, USA).