Coomassie brilliant blue r solution

Spent saliva from each disc at each time point was centrifuged at 16,000 RPM for 5 minutes. The supernatant was saved and split into two aliquots; one sample for protein analysis and one for metabolic analysis. A 15 μl aliquot of sample was added to 5 μl of loading dye and 1 μl dithiothreitol, and 15 μl of this was loaded onto a 4–12% Bis-Tris ready-made polyacrylamide gel (Invitrogen, USA) and run for 32 minutes at 200 volts constant and 120 amps for one gel, 240 amps for two gels. Gels were stained with Coomassie brilliant blue R solution (Merck, Germany) for 20 minutes and de-stained in 10% acetic acid. Gel images were captured using the ChemiDoc (Bio-Rad, USA) and images were analysed using Image Lab (Bio-Rad) using relative quantity tools.

Three spent saliva samples from each disc per treatment were centrifuged at 16,000 × g for 5 min. A 15 μl aliquot of supernatant was added to 5 μl of loading dye and 1 μl of dithiothreitol, and 15 μl of this was loaded onto a ready-made 4–12% Bis-Tris polyacrylamide gel (Invitrogen, USA), run for 32 min at 200 volts constant, stained with Coomassie brilliant blue R solution (Merck, Germany) and de-stained with 10% acetic acid. A ChemiDoc (Bio-Rad, USA) was used to capture gel images, which were analyzed using Image Lab (Bio-Rad) using protein volume intensity tools (whole lane and protein band).