Protean xi

Each saliva sample was run in duplicate. For each sample, a volume corresponding to 6.5 µg total protein was mixed with sample buffer and run on each lane of a 14% polyacrylamide mini-gel (Protean xi, Bio-Rad, CA, USA) using a Laemmli buffer system, as described elsewhere [19 (link)]. An electrophoretic run was performed at a constant voltage of 140 V until front dye reached the end of the gel. Gels were fixed for 1 h in 40% methanol/10% acetic acid, followed by staining for 2 h with Coomassie Brilliant Blue (CBB) G-250. Gel images were acquired using a scanning Molecular Dynamics densitometer with internal calibration and LabScan software (GE Healthcare, Chicago, IL, USA), and images were analyzed using GelAnalyzer software (GelAnalyzer 2010a by Istvan Lazar, www.gelanalyzer.com, assessed on February 2020) for the normalized volume (volume percentage) of each protein band. Molecular masses were determined in accordance with molecular mass standards (Bio-Rad Precision Plus Protein Dual Colour 161–0394) run with protein samples.

Salivary proteins were first separated according to molecular masses using SDS-PAGE. Each saliva sample was run in duplicate, considering, for each of them, a volume corresponding to 7 µg of total protein. This volume was mixed with sample buffer and run on each lane of a 14% polyacrylamide mini-gel (Protean xi, Bio-Rad, Hercules, CA, USA) using a Laemmli buffer system, as previously described [26 (link)]. The electrophoretic run occurred at a constant voltage of 140 V, at room temperature, until the front dye reached the end of the gel. Gels were fixed for 1 h in 40% methanol/10% acetic acid, followed by staining for 2 h with 2% Coomassie Brilliant Blue (CBB) R-250 and destaining in several washings of 10% acetic acid. The gel images were acquired using a scanning Molecular Dynamics densitometer with internal calibration and LabScan software (GE Healthcare, Chicago, IL, USA), with images analyzed using the GelAnalyzer software (GelAnalyzer 2010a by Istvan Lazar). Molecular masses were determined in accordance with molecular mass standards (Bio-Rad Precision Plus Protein Dual Colour 161–0394) run with protein samples.

Due to the possible different effects, of each inactivation protocol, in the results obtained from total protein assays^{17 (link)}, the total protein concentration of controls (TT1) was used for calculation of the volume to be used in SDS-PAGE runs. Each saliva sample was run in duplicate and, for each, a volume corresponding to 7.5 µg total protein was mixed with sample buffer and run on each lane of 14% polyacrylamide mini-gels (Protean xi, Bio-Rad, CA, USA) using a Laemmli buffer system, as described elsewhere^{2 (link)}. In each gel, one of the lanes was used for molecular mass standard (Bio-Rad Precision Plus Protein Dual Colour 161-0394) running. The electrophoretic run was performed at a constant voltage of 140 V until the front dye reached the gel's end. Gels were fixed for 1 h in 40% methanol/10% acetic acid, followed by staining for 1 h with Coomassie Brilliant Blue (CBB) R-250 and destained in several washes of 10% acetic acid. Gel images were acquired using a scanning Molecular Dynamics densitometer with internal calibration and LabScan software (GE Healthcare), and images were analyzed using GelAnalyzer software (GelAnalyzer 2010a by Istvan Lazar, https://www.gelanalyzer.com) for the volume percentage of each protein band.

Proteins of interest (fractions eluted after VVA or R#438 affinity chromatographies) were separated after SDS-PAGE on a vertical slab gel system (0.1 × 16 × 14 cm; Protean Xi, BioRad). Gels were run at 15 mA/gel during migration in a stacking gel and at 27 mA/gel in the separating gel (12%). Molecular weight standards (LMW Electrophoresis calibration Kit, Amersham Bioscience, Uppsala, Sweden) were run simultaneously. Proteins were transferred onto 0.2 μm polyvinylidene difluoride (PVDF) membranes (BioRad) for N-terminal microsequencing. The transfer was carried out on a Transblot Cell Apparatus (BioRad) at a constant voltage (60 V) during 2 h 30 min. The PVDF membrane was stained with 0.2% (w/v) Coomassie Brilliant Blue R-250 for 5 min and destained 3 times in 50% methanol solution. After PVDF membranes were dried, proteins were excised and subjected to Edman degradation on a pulsed liquid-phase protein sequencer (Procise 492 Applied Byosystems, Foster City, CA, USA).
The N-terminal sequences obtained in fallow deer have been deposited in the EMBL-EBI database (Swiss-Prot: C0HJC7, C0HJC8, C0HJC9, C0HJD0). N-terminal sequences were compared to those described in databank in order to detect homologies with other isolated native proteins or those deduced from cDNA (Blast, NCBI).