One-dimensional SDS-Polyacrylamide Gel Electrophoresis (1D SDS-PAGE)
Samples at different dilutions or BSA standards at different concentrations were mixed with 5 mM DTT and NuPAGE LDS sample loading buffer (4×) to have a final concentration of 0.5-1 mM DTT and 1× sample loading buffer. The mixture was then heated at 85°C for at least 5 mins to denature proteins. Electrophoresis was performed using a NuPAGE 4-12% Bis-Tris gel (1.0 mm×15 well, Invitrogen) according to the manufacturer’s instructions and the gels were run using 1×MES running buffer from a 20× stock (Thermo Fisher Scientific, Somerset, NJ). PageRuler Plus Pre-stained Protein Ladder from Thermo Fisher Scientific (Somerset, NJ) was used as molecular weight marker. Gel was run at 200 V for 35 min and later stained either with Imperial staining solution or Mass Spectrometry compatible silver staining kit (Pierce, Thermo Scientific, Somerset, NJ) according to the manuals provided in the kit.