Bio safe coomassie

Cells were collected at four different time points during fermentation: 0, 24, 48, and 72 h. Cell pellets were re-suspended in an sodium dodecyl sulfate (SDS) lysis buffer containing 4% SDS in 100 mM triethylammonium bicarbonate pH 8.0, 10 mM ethylenediaminetetraacetic acid and a protease inhibitor cocktail (Roche). The cell extract was boiled at 90°C for 10 min followed by sonication for 30 s. The cell debris was removed by centrifugation. The protein pellet obtained after chloroform/methanol precipitation was dissolved in denaturation buffer containing 8 M urea in 10 mM Tris–HCl pH 8.0. Protein concentration was measured by Bradford protein assay. A total of 100 μg of each sample were separated on a Mini-Protean^® TGX^TM 4–20% gradient gel (Bio-Rad) and stained with Bio-Safe^TM Coomassie (Bio-Rad). Regions corresponding to the size of “DhaB1” were cut and in-gel trypsin (Pierce^TM) digestion was performed as described (Shevchenko et al., 2007 (link)). Peptides were desalted using C-18 stage-tips and analyzed on the Orbitrap Fusion^TM Tribrid^TM (Thermo Fischer Scientific). An inclusion list consisting of m/z values in the range of 400–1600 Da and charge states +2 and +3 only was incorporated into the instrument method. Acquired mass spectra were processed with the MaxQuant software suite (v.1.5.1.0; Cox et al., 2009 (link)).

The flow-through, wash fractions and elution fractions were separated by 10% or 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE) followed by protein staining using Bio-SafeTM Coomassie (Bio-Rad, Hercules, CA, USA) according to manufacturer’s instructions or transferred to nitrocellulose membranes (Schleicher and Schull, Dassel, Germany) for western blot analysis. In comparative experiments, run in parallel, the same amounts were loaded on the gels, for each parallel fraction. Non-specific sites on the nitrocellulose membranes used for western analysis were blocked in a blocking solution (10 mM Tris pH 8, 150 mM NaCl, and 0.05% Tween 20 [TBST]) supplemented with 5% non-fat dried milk (1 hour, at room temperature). The Protein complexes were visualized by SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA).and exposed on Fuji Film Medical X-ray film (Fuji Corporation, Tokyo, Japan).

SulfoBiotics-Protein Redox State Monitoring Kit (Dojindo) was used to label the reduced (free) Cys residues of mGal-1 WT and mGal-1/Ox proteins. The labeling of the reduced Cys residue with Protein-SHifter was performed according to the manufacturer’s instructions. The labeled proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and coomassie brilliant blue staining using Bio-Safe™ Coomassie (Bio-Rad, Hercules, CA, USA). The redox state of Cys residues was monitored based on molecular weight because the labeling of one Protein-SHifter molecule resulted in a molecular shift of approximately 15 kDa.