Nzyblue protein marker

All chemicals were of an analytical grade. Water, methanol (MeOH), formic acid, and acetonitrile (LC/MS grade Optima), chloroform, Pierce™ Trypsin Protease MS Grade, Pierce™ DTT (Dithiothreitol), Bolt^® MOPS SDS Running Buffer (20×), mini protein gel NuPAGE™ 4 to 12% Bis-Tris, and 4X Bolt™ LDS Sample Buffer were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), trypsin, glutamine, phosphate-buffered saline (PBS), and Foetal Bovine Serum (FBS) were obtained from Lonza^® (Verviers, Belgium). Ethanol (96%) was purchased from Carlo Erba (Peypin, France). Iodoacetamide and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Barcelona, Spain). Tris(hydroxymethyl)aminomethane and glacial acetic acid were obtained from Merck Milipore^® (Burlington, MA, USA, EUA). Coomassie Brilliant Blue R-250 was purchased from BIORAD^® (Hercules, CA, USA). A 5× SDS-PAGE Sample Loading Buffer and NZYBlue Protein Marker were purchased from Nzytech^® (Lumiar, Portugal).

After casting, gels were placed inside an electrode assembly tank and afterward inside a tank. Both chambers were filled with Running Buffer 1× (25 mM Tris, 192 mM glycine, 0.1% (v/v) SDS, pH 8.3). Prior to electrophoresis, previously precipitated samples and the remaining agarose beads, were mixed with 30 μL of sample buffer (62.5 mM Tris-HCl pH 6.8, 25% (v/v) glycerol, 2% (v/v) SDS, 5% (v/v) β-mercapthoetanol and 0.01% (w/v) bromophenol blue) and boiled for 5 min at 96 °C. Agarose beads required an extra 4 min centrifugation at 15,000× g to obtain the supernatant fraction (non-eluted fraction—NE). Each gel was loaded with 5 μL of molecular weight markers (NZYBlue Protein marker, NZYTech) and 30 μL of each pulldown fraction. The run was performed in a mini-PROTEAN Tetra System (Bio-Rad, Hercules, CA, USA) at 50 V. After glycoproteins separation by molecular weight, gels were transferred to a PVDF membrane to assess for the presence of glycoproteins by lectin-blotting.