SDS-PAGE and Immunoblotting of T. marneffei Proteins

Twenty micrograms/lane of TM CYA, concentrated culture supernatant, or T. marneffei EVs were mixed with Laemmli sample loading buffer containing β - mercaptoethanol and heated in boiling water for 10 min. The mixtures were subjected to SDS-PAGE separation on a reducing pre-cast Novex WedgeWell 4–20% Tris-glycine gel (Invitrogen, Carlsbad, CA, USA). Protein bands were stained with Commassie InstantBlue (Expedeon, Cambridge, UK). For immunoblotting, the polyacrylamide gel containing separated polypeptides were transferred electrophoretically to the 0.45 µm nitrocellulose membrane (Amersham, Uppsala, Sweden). Immunoblotting was performed using the T. marneffei yeast phase specific mannoprotein MAb 4D1 and melanin binding MAb 8D6 as described (30 (link)).