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Forty micrograms of the respective Ovophis venoms were electrophoresed with 15% SDS-PAGE under reducing conditions in accordance with the Laemmli protocol [53 (link)], calibrated with the prestained protein ladder (5–245 kDa). The electrophoretic separation was conducted at 90 V for 120 min. Proteins were stained with Coomassie Brilliant Blue R-250 for visualization. The gel was then scanned using Image Scanner III Labscan 6.0 (GE Healthcare, Chicago, IL, USA). The relative intensity of gel regions (I, II, and III) were determined using myImage analysis software (Thermo Scientific, Waltham, MA, USA).

Naja nivea venom and the RP-HPLC-collected fractions were reconstituted in ultrapure water and separated by 15% SDS-PAGE under reducing conditions at 100 V for 2 h. Spectra™ Multicolor Broad Range Protein Ladder (10–260 kDa) was used for molecular mass calibration. Gels were stained with Coomassie Brilliant Blue R-250 and scanned using Image ScannerIII Labscan 6.0 (GE Healthcare, Uppsala, Sweden).

Silicone catheter pieces (1 or 2 cm) were incubated in 6-well polystyrene tissue culture plates containing 5 ml of DMEM+FBS that had been inoculated with S. epidermidis strains to an OD₆₀₀ of approximately 0.05. After 2 h at 37°C, cells were exposed to 1 mM of DETANONOate and 5 mM of oxamate and incubated for 22 h. The images of the silicone sections were recorded with a digital camera. Then, the media were removed, and the plates were placed at 65°C for 2 h. The silicone pieces in the wells were submerged in 5 ml of 1% crystal violet for 20 min, washed and rinsed twice with PBS (1×), and air-dried. Images of the wells were acquired in an ImageScanner III LabScan 6.0 (600 dpi, GE Healthcare). The silicone tubes were then transferred to falcon tubes and destained by 10-min incubation with 5 ml of glacial acetic acid (33%), after which the CV absorption was measured at 590 nm.

Stained gels were scanned, and the images were acquired by Image Scanner III LabScan 6.0 (GE HealthCare, Chicago, IL, USA). The 2D gel image analysis software PDQuest (Bio-Rad, Hercules, CA, USA) version 7.2.0 was used for gel-to-gel matching and identifying differences in the proteomes. Each of the three pH-specific sample sets was analyzed using three independent biological replicates on three different 2D gels. The gel images were normalized in the PD Quest software to even out differences in staining intensities between gels. Each matched protein spot was assigned a unique sample spot protein (SSP) number in the PD Quest software. For gel comparison, a statistical approach was applied when determining differentially expressed proteins using the PD Quest software. The Student’s t-test was performed using a 95% significance level to determine which proteins were differentially abundant between experimental and controls tissues. The differentially abundant proteins were identified by MALDI TOF/MS and LC-MS/MS.

SDS-PAGE electrophoresis was conducted by a standard procedure by Laemmli [39 (link)]. The procedure was carried out in a 10% resolving gel (Tris–HCl buffer with pH 8.8), and 4% polyacrylamide in Tris–HCl buffer with pH 6.8 was used as a stacking gel and Tris–glycine running chamber buffer. Separation took place at a constant current of 100 mA. Additionally, a molecular weight standard (Perfect TM Color Protein Ladder, EurX, Gdańsk, Poland) with a molecular weight range from 7 to 240 kDa was used during separation. Gels were stained with Coomassie blue (Sigma-Aldrich, Poznań, Poland) and silver. The stained gels were scanned by ImageScanner III LabScan 6.0 (GE Healthcare, Chicago, IL, USA).