Benchmarktm protein ladder

For 1-DE gel electrophoresis, samples of total extract and acetonic fraction of B. pilosa were assessed by 12% polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) in non-reducing conditions (25 (link)), in parallel with molecular weight markers (BenchMarkTM Protein Ladder 6–200 kDa, Invitrogen, Karlsruhe, Germany). Gels were stained with Coomassie brilliant blue G-250® (Sigma-Aldrich, St. Louis, MO, USA). The molecular weight bands were estimated by linear regression analysis, based on the calculation of relative mobility (Rf), using KODAK 1D Image Analysis program (Eastman Kodak Co., Rochester, USA). Additionally, the band was submitted to MS/MS and identified in the National Center for Biotechnology Information (NCBI) database.
For 2-DE gel electrophoresis, 60 μg of acetonic fraction were diluted in ultrapure water and separated by isoelectric focusing (IEF) on 7-cm immobilized pH gradient strips (ReadyStripTM IPG Strip pH 3–10) overnight at room temperature, following the manufacturer instructions (GE, Healthcare, Uppsala, Sweden). After IEF, strips were equilibrated and running onto precast 12% polyacrylamide gels, being the staining spots analyzed by ImageMasterTM 2-D Platinum 7.0 (GE Healthcare, Amersham Pharmacia Biotech, United Kingdom) to be submitted to MS/MS and identified in the NCBI database.

The changes at the proteome level in the plant–fungus interaction were studied using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE). For SDS-PAGE, 250 µg/µL protein samples were loaded and run using Mini-PROTEAN Tetra Cell apparatus at 90 V (Bio–Rad, Hercules, CA, USA) in 12% gels with a molecular weight marker (Bench Mark^TM Protein ladder, Life Technologies, Bangalore, India). Gels were stained using a solution of 0.2% (w/v) Coomassie Brilliant Blue R250 (Sigma-Aldrich, St. Louis, MO, USA), methanol (Sigma-Aldrich, St. Louis, MO, USA) 40% (v/v) and acetic acid (Sigma-Aldrich, St. Louis, MO, USA) 10% (v/v) for 90 min and destained with a solution of methanol 40% (v/v) and acetic acid 10% (v/v) for 90 min and later stored in acetic acid 5% (v/v) at room temperature.