Tgx precast protein gel

Cells were lysed with 10% cell lysis buffer (Cell Signaling Technology, Denver, CO, USA) and 0.005% phenylmethylsulfonyl fluoride (PMSF) (Cell Signaling Technology), and then the protein lysates (10 μg) were separated by 10% polyacrylamide gel electrophoresis (TGX precast protein gels, Bio-Rad, Hercules, CA, USA). Green fluorescent protein (GFP) and β-actin proteins were detected by western blotting, which was performed in accordance with a protocol published previously (11 (link),30 ). GFP fold change was calculated from the band intensity of GFP after normalization against the band intensity of an internal control (β-actin). All calculations were performed by using ImageJ software (https://imagej.nih.gov/ij/), as described in references (11 (link)) and (30 ).

Whole-cell and cellular fractions were lysed in Laemmli sample buffer (125 mM Tris (pH 6.7), 20% (v/v) glycerol, 140 mM SDS, 0.3 µM bromophenol blue) supplemented with 0.1 M DTT, cOmplete™, Mini Protease Inhibitor Cocktail (Roche, 4693124001) and PhosSTOP™ (Roche, 4906837001). Lysates were boiled, and proteins were separated by SDS-PAGE using a 4%–15% TGX™ Precast Protein Gel (BioRad, 4561086/5671085) and blotted onto a nitrocellulose membrane (BioRad, 1704158/59) using a Trans-Blot TurboTM transfer system. Novex Sharp Protein Standard (Thermo Fisher Scientific LC5800) was used to evaluate the molecular weights of proteins from the gels. Following blocking in PBS containing 0.1% (v/v) Tween-20 (PBST) and 5% (w/v) BSA, membranes were cut in appropriate sizes for detection of proteins of interest and incubated ON at 4°C with primary antibodies in 0.1% (v/v) NaN₃/5% (w/v) BSA/PBST (Supplementary Table S1). Next, membranes were incubated for 30 min with matching secondary HRP-conjugated antibodies (Supplementary Table S1) and the immunoreactivity was detected using a luminescent image reader (Fujifilm, LAS-4000) following incubation with Clarity™ Western enhanced chemiluminescent substrate (BioRad, 1705061).

For growth experiments in different carbon source-supplemented media, B-medium was supplemented with 0.5% (w/v) AX, 0.5% (w/v) crystalline chitin (CHI), 0.5% (w/v) XG, 0.2% (w/v) heat-treated C.vulgaris biomass (C.v.) and 0.2% (w/v) C.vulgaris AIS (C.v. AIS). B-medium without any carbon source (NS) was used as negative control. Chitin from shrimp shells was purchased from Sigma-Aldrich (Saint Louis, USA). All polysaccharides were sterilized and supplied to B-medium (pH 6.5). Filtrates from culture media were prepared and assayed according to the procedure previously described. Alternatively, the filtrates were separated in a TGX™ Precast Protein Gel [4–15% (w/v) polyacrylamide] (Biorad, CA, USA) using Precision Plus Protein™ Dual Color Standards (Biorad, CA, USA) as molecular weight marker and then stained by silver nitrate. Proteolytic assay was performed by incubating 1 µg BSA with 20 µL of C.v.-filtrate for 16 h at 28 °C. BSA was purchased from Sigma-Aldrich (St. Louis, USA). The reaction was separated in 10% of Laemmli gel using Precision Plus Protein™ Dual Color Standards (Biorad, CA, USA) as molecular weight marker and then stained by silver nitrate.