Novex bolt 4 12 bis tris plus gel

The protein concentration was measured by the Qubit Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used for the separation of proteins. Samples (10 µg of proteins) were loaded and electrophoretically separated on a Novex Bolt 4–12% Bis-Tris Plus gel (Invitrogen, Carlsbad, CA, USA), using Bolt MOPS SDS running buffer (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The gel was stained with colloidal Coomassie Brilliant blue G-250 (AppliChem, Darmstadt, Germany) overnight and rinsed with MilliQ water until the background became clear.

Protein profiles of the different vesicle isolates were obtained by SDS-PAGE. Samples (10 μg of protein based on Qubit protein assay) were electrophoretically separated under reducing conditions on a precast Novex Bolt 4–12% Bis-Tris Plus gel (Invitrogen, Carlsbad, CA, USA) using the Bolt MOPS SDS running buffer (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Gels were stained with colloidal Coomassie blue (Applichem GmbH, Darmstadt, Germany).

Tomato fruits (Piccadilly variant) were purchased in the local market (G.M Fruit, Sicily, Italy). Sodium hydrogen phosphate dihydrate (Na₂HPO₄·2H₂O), sodium dihydrogen phosphate (NaH₂PO₄·H₂O) and sodium hydroxide (NaOH) were from J.T. Baker (Deventer, The Netherlands). Leupeptine, phenylmethylsulfonyl fluoride (PMSF), sodium azide, sucrose and colloidal Coomassie Brilliant blue G-250 were obtained from AppliChem (Darmstadt, Germany). OptiPrep™ (60% (w/v)) was from Serumwork (Bernburg AG, Germany). Sepharose Cl-2B, sucrose and Triton^® X-100 were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). RapiGest detergent was obtained from Waters Corporation (Milford, MA, USA). Trypsin (Mass Spec grade) was from Promega Corporation (Madison, WI, USA). Qubit Protein Assay Kit was from Thermo Fisher Scientific (Rockford, IL USA). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) buffers, reagents and materials: Novex Bolt 4–12% Bis-Tris Plus gel and Bolt MOPS SDS running buffer were from Invitrogen (Carlsbad, CA, USA). Water (18.2 MΩ.cm (25 °C), 0.22 µm filtered) generated by a MilliQ system (Merck Millipore) was used. Other solvents used for proteomics were LC–MS grade (VWR International, Debrecen, Hungary). Leucine enkephalin peptide (amino acid sequence is YGGFL) was purchased from Waters Corp. (Wilmslow, UK).