Anti gapdh ga1r

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).

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: M75⁴⁷ (link), anti-Tsg101 (4A10, GeneTex, USA), anti-GAPDH (GA1R, Santa Cruz Biotechnology, USA) and anti-actin (C4, Santa Cruz Biotechnology, 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).