Protein extraction using SDS-DTT buffer

Protein extraction was performed according to a previously described method [24] with minor modifications. Dried biomass (50 µg) was incubated in 5 mL of SDS-DTT buffer for 10 min at 80 °C to activate the protein extraction and then left rotating at 40 °C for 2 h. After 2 h, the samples were centrifuged at 3220 ×g for 20 min at room temperature. Then the protein-containing supernatant was collected. Ice-cold acetone was used to precipitate the protein from the supernatant. The precipitate was incubated at − 20 °C overnight. On the next day, the samples were centrifuged at 16,000 ×g for 20 min at 4 °C in a bench top 5415R centrifuge (Eppendorf, Hamburg, Germany), and the pellet was re-suspended in 50 mM Tris–HCl buffer (pH of 8.0). The acetone precipitation of the protein was repeated twice. Subsequently, the protein pellet was either re-solubilized in 1 mL of 50 mM Tris–HCl, pH 8.0, and then directly used in SDS-PAGE analysis or was re-solubilized in 1 mL of rehydration buffer (2 M thiourea, 6 M urea, 16.2 × 10⁻³ M 3-[(3-Cholamidopropyl) dimethyl ammonio]-1-propane sulphonate, 25.9 × 10⁻³ M DTT) supplemented with ampholytes (BioRad, Munich, Germany) according to manufacturer’s specification and used for 2D-PAGE analysis.