Bradford protein assay

Protein concentration was performed using Bradford protein assay (Life Technologies, Carlsbad, CA, #23236) following the manufacturer’s protocol.

The fractions obtained of the SEC were tested using a Bradford protein assay (Life Technologies, Carlsbad, CA, USA) to acquire protein elution profiles. Each sample was diluted 1 : 3 in PBS 1× and incubated for 10 minutes with the Bradford reagent (1 : 1) at room temperature. Measurements were performed in duplo in Costar 96 well plate (Sigma-Aldrich, St. Louis, USA) and repeated three times. OD values were normalized using a negative control and converted to concentrations (μg/mL) using an albumin standard curve (R² = 0.996) (Life Technologies, Carlsbad, CA, USA).

Strains were cultivated as detailed above, and cells were then collected hourly by centrifugation and disrupted using Fast Prep (FP120 Thermo Savant). Cell debris in the supernates was discarded after centrifugation. The cell-free lysates were then utilized to measure the enzymatic activity of LDH and GPDH after suspension in 100 µL LDH assay buffer (Sigma-Aldrich MAK066 Kit, Burlington, VT, USA). Activities of LDH and GPDH were determined from three independent biological replicates. Protein concentrations were measured using the Bradford protein assay (Fisher Scientific, Waltham, MA, USA).

Protein concentration was determined by a modified Bradford protein assay (Fisher Scientific)8 (link). Protein size distribution was analysed on an Experion automated electrophoresis system using Pro260 chips (Bio-Rad, Marnes La Coquette, France).

Bacteria were cultivated as described; bacteria were harvested hourly by centrifugation and disrupted by using a Fast Prep instrument (FP120 Thermo Savant). Cell debris was removed by centrifugation. The cell-free lysate was used for the LDH activity assays (Sigma-Aldrich MAK066 Kit). LDH activity was measured from three biological replicates. Protein concentrations were determined by using the Bradford protein assay (Fisher Scientific, Waltham, MA, USA).

Concentration of HIF-1 α protein in cell culture lysates was quantified using the quantitative sandwich ELISA immunoassay (Invitrogen, HIF-1 α ELISA Kit; EHIF1A). HIF-1α standards and samples were captured by a polyclonal HIF-1α antibody on the pre-coated plate. Detection was then performed using a biotinylated monoclonal HIF-1α antibody reactive to epitopes other than the capture antibody. All reagents, samples and standards were prepared as instructed in the manual. Prior to ELISA, the cells were treated with different levels of oxygen as previously described. The cell lysate was collected in cultured adherent cells to approximately 80% confluence on T25 cell culture flask (Invitrogen). The cells were then scraped using a cell scraper and the lysate transferred to a 15 mL conical tube. The Bradford protein assay was used to measure the concentration of total protein for each sample (Invitrogen, 23246). All the experiments were conducted in triplicate.