Molecular dynamics densitometer

Samples from 33 females (20 sensitive and 13 low-sensitive) and 41 males (19 sensitive and 22 low-sensitive) were subjected to SDS PAGE. Each sample was run in triplicate. A total of 7.5 ug protein from each individual saliva sample was run on each lane, in a 12% polyacrylamide mini-gel (Protean xi, Bio-Rad), using a Laemmli buffer system [15]. An electrophoretic run was performed at a constant voltage of 150 V 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 Coomassie Brilliant Blue (CBB) G-250. Gel images were acquired using a scanning Molecular Dynamics densitometer with internal calibration and LabScan software (GE Healthcare), and images were analysed using Gel Analyzer software (GelAnalyzer 2010a by Istvan Lazar, www.gelanalyzer.com). Molecular masses were determined in accordance with molecular mass standards (Bio-Rad Precision Plus Protein^TM Dual Color 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.