Amicon ultra 0.5 ml centrifugal 3 kda filters

We also tested if the addition of biosurfactants can cause extraction of proteins from the C. albicans cell surface. To conduct the experiment, Candida cell suspensions in PBS were transferred to Eppendorf test tubes and biosurfactants were added to the final concentrations. The same amount of PBS was added to the control samples. Suspensions were incubated for 2 h at 37 °C with agitation (300 rpm). Then cells were removed by centrifugation (1000×g) and filtration (0.2 µm). Proteins in supernatants were concentrated with Amicon Ultra 0.5 mL 3 kDa centrifugal filters (Millipore, USA). Concentrated samples were mixed with ×6 denaturation buffer (150 mM Tris; 0.6 M EDTA; 12 % SDS; 60 mM DTT), heated at 95 °C for 5 min and loaded onto 15 % polyacrylamide gel. Silver-stained gels were photographed with ChemiDoc System (Bio-Rad, USA).

Protein samples (control and different grades of meningiomas) in rehydration solution were exchanged to TEAB buffer using Amicon Ultra 0.5 mL centrifugal 3 kDa filters (Millipore, Watford, UK). Similar sample pooling strategy for control and different grades of meningiomas, which was used for 2D-DIGE analysis, was followed. After buffer exchange, quantification of protein content in each sample was performed using QuickStart Bradford reagent (BioRad, USA). Prior to the iTRAQ labeling, in-solution digestion was performed (75 μg proteins from each sample) following the manufacturer's instructions. trypsin (trypsin Gold, mass spectrometry grade, Promega, Madison, WI, USA) was used at a 1:20 trypsin: protein ratio. After in-solution digestion, iTRAQ labeling of the peptides was performed following the manufacturer's instructions (AB Sciex UK Limited, UK). Following labeling strategy was implemented for differential proteomic analysis; control-114, MGI-115, MGII-116 and MGIII-117. All the labeled samples were pooled and concentrated using a speed vac. OFFGEL fractionation of the labeled peptides was performed using a 3100 OFFGEL fractionator (Agilent Technologies, Santa Clara, CA) with high resolution (pH 4–7, 24 cm) IPG strips.

Proteins in rehydration buffer (described earlier) were exchanged to 0.5 M TEAB buffer (compatible for iTRAQ labeling) using Amicon Ultra 0.5 mL centrifugal 3 kDa filters (Millipore, Watford, UK). Following buffer-exchange protein concentrations were determined using QuickStart Bradford reagent (BioRad, USA) and quality was checked on a 12% SDS gel. In-solution digestion of respective protein samples (100 μg each); control (0 h) and treatments (40 h, 88 h, and 120 h) were performed using trypsin (trypsin Gold, mass spectrometry grade, Promega, Madison, WI, USA) at a ratio 1: 30 (trypsin: protein), following the manufacturer’s instructions. Four-plex iTRAQ labeling kit (AB Sciex UK Limited, UK) having labels 114, 115, 116 and 117 was used to label trypsin-digested peptides of 0, 40, 88 and 120 h FC2 samples respectively, following the manufacturer’s instructions. All the labeled peptides were pooled and proceeded for OFFGEL fractionation using a 3100 OFFGEL Fractionator (Agilent Technologies, Santa Clara, CA) with high resolution (pH 3–10, 24 cm) IPG strips. Fractions were collected and enriched using Zip-Tip C18 pipette tips (Millipore, USA).

Serum samples from each of the study cohorts (HC, NSVM and SVM) were divided into three separate pools (n = 10) for iTRAQ-based quantitative proteomics analysis. Protein samples (in rehydration solution) were exchanged to TEAB buffer using Amicon Ultra 0.5 mL centrifugal 3 kDa filters (Millipore, Watford, UK). After buffer exchange, quantification of protein content in each sample was performed using QuickStart Bradford reagent (BioRad, USA). In-solution digestion (75 μg proteins from each sample) and subsequent iTRAQ labeling of the digested peptides were performed following the manufacturer’s instructions (AB Sciex, USA). HC samples were labeled with the 114 iTRAQ reagent, while NSVM and SVM samples were labeled with 115 and 116 iTRAQ labels, respectively. OFFGEL fractionation of the labeled peptides was performed using a 3100 OFFGEL fractionator (Agilent Technologies, Santa Clara, CA) with high resolution (pH 4-7, 24 cm) IPG strips. In-solution digestion, iTRAQ labeling, and OFFGEL fractionation protocols are described in details elsewhere42 (link).