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.