Pdquest 8

IPG strips in 2-DE were used as previously reported^{42 (link), 43 (link)}. Briefly, 17-cm IPG strips (pH 3–10) (Sigma-Aldrich) were rehydrated actively at 50 V, and urine protein samples (3 mg/strip) were placed in rehydration buffer with 0.002% (w/v) bromophenol blue for 16 h at 20 °C in a protean isoelectric focusing (IEF) cell (Bio-Rad, Hercules, CA, USA). IEF was performed under the following conditions: 150 V for 3 h, 300 V for 3 h, 1000 V (gradient) for 4 h, 10,000 V (gradient) for 5 h, and 10,000 V up to 60 kV-h. The strips were equilibrated in 10 mL of equilibration buffer (65 mM DTT in 6 M urea, 50 mM Tris-HCl, pH 8.8, 30% v/v glycerol, 2% w/v SDS, and 0.001% v/v bromophenol blue) for 15 min and transferred to 10 mL of equilibration buffer containing 135 mM iodoacetamide instead of dithiothreitol. The equilibrated IPG strips in 0.5% low-melting point agarose were placed on top of a 10% acrylamide separating gel (pH 8.8) and run at a constant voltage of 100 V at 20 °C using the Protean II xi multi-cell with a 2-DE conversion kit (Bio-Rad) until the dye front reached the end of the gel. Each experiment was independently performed twice. The stained gels were scanned using a GS-800 calibrated densitometer (Bio-Rad, Hercules, CA, USA), and the images were exported to PD Quest 8.0 software (Bio-Rad, Hercules, CA, USA) for image analyses.

A 12% acrylamide gel was prepared; loading amount was 20 μL, containing the volume ratio of protein sample and 5× protein loading buffer (4:1). Before gel loading, the protein sample was heated to 100°C for 5 min in a water bath.
The loading volume was determined as 150 μL containing 200–250 μg total protein, and protein was diluted with hydration buffer to 150 μL (Zhang et al., 2015 (link)). The loading volume was calculated according to the results of protein qualitation. The strips were taken out from the refrigerator at −20°C and placed at room temperature for 1 h. The processed samples were put into the swelling tank, which was covered by the strips with the addition of mineral oil. Isoelectric focusing (IEF) was performed in PROTEAN Isoelectric Focusing System (Bio-Rad, USA). The temperatures of hydration and IEF were both set at 20°C. The hydration time was 12 h. The procedures of IEF are detailed in Table 1. Then, vertical SDS-PAGE electrophoresis was performed. After staining with Coomassie brilliant blue R-250 for 20 min, appropriate amount of decoloration solution was added (absolute alcohol:glacial acetic acid:water = 1:1:8). Decoloration was performed on a silent shaker, and a GS-800 Calibrated Densitometer was used for scanning. The PDQuest 8.0 software (Bio-Rad, USA) was used to identify proteins that were expressed differentially.

A protein load of 100 μg (analytical gels) and 300 μg (preparative gels) of the urea/chaps serum extracts was applied to 17-cm dry strips (pH 4–7 linear range; BioRad). The second dimension was resolved in 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Gels were developed by fluorescent staining (Flamingo; BioRad, Hercules, CA, USA). For each independent condition, 2-DE for protein extracts from the baseline and the post-intake of placebo or Fruit-Up^® were processed in parallel to guarantee maximum comparability. Analysis for differences in the protein profiles was performed with the PD-Quest 8.0 software (BioRad, Hercules, CA, USA). Each spot was assigned a relative value that corresponds to the single spot volume compared to the volume of all the spots in the gel, following background extraction and normalization between gels [26 (link)].

The gels were scanned using a VersaDoc4000 image system (Bio-Rad) and the images were analysed with PDQUEST 8.0 software (Bio-Rad, USA). There were three biological replicates per treatment with at least three gels for each biological replicate. Only spots with a variation rate of ±0.5 in the three replicates were considered for further analysis. Stained protein spots were excised manually from the gels, in-gel digested with trypsin, and analysed using a MALDI-TOF/TOF mass spectrometer (ABI 4800). The MASCOT database search engine (http://matrixscience.com) was used to search for peptide mass lists from the obtained spectra against the NCBI database. The mass error tolerance was set to 80 ppm, and the score threshold was above or equal to 110.

Proteins were identified by the peptide fingerprint search with Mascot software (Matrix Science Inc., Boston, MA, USA) through the Sb. thermotolerans Kr1 protein database (accession number CP019454; https://www.ncbi.nlm.nih.gov/nuccore/CP019454.1?report=genbank). Protein scores greater than 45 were considered significant (p < 0.05). The quantitative assessment was performed with the PDQuest 8.0 software (Bio-Rad, Hercules, CA, USA). Differentially expressed proteins with a fold change of ≥2.0 were discussed.

Gels were stained with Coomassie Brilliant Blue G-250 overnight, rinsed with deionized water, and scanned using a PowerLook 2100XL scanner (UMAX, Xinzhu, Taiwan). The gel images were analyzed by PDQuest 8.0 software (Bio-Rad).

Chemical and mineral analyses were carried out in duplicate with three replicates (ore concentrate) and in two experiments with three samples and three series of measurements for each sample (elements in cells and the liquid phase). Two series of experiments on the bacterial growth included three parallels (flasks) and three replicates for each measured parameter. Statistical processing was performed using Microsoft Excel 2010. The standard deviation (SD) of the arithmetic mean was calculated, and the significance of the results was assessed using the Student’s t-test at the significance level p ≤ 0.05. Two series of proteomic experiments under different growth conditions and three protein extractions (biological replicates) with three or four 2D gels for each extraction were carried out. Data were statistically processed using the PDQuest 8.0 software (Bio-Rad, Hercules, CA, USA). The means of three biological replicates in two experiments (±SD) were discussed.