Instantblue protein gel stain

Unphosphorylated, single phosphorylated and double phosphorylated KaiC_RS were separated by 10% SDS–PAGE with 37.5:1 acrylamide:bis-acrylamide (Bio-Rad), 18 cm × 16 cm × 1 mm Tris-HCl gel with 1× Tris-glycine SDS running buffer (Invitrogen). The samples were heated at 95 °C for 3 min, and 400 ng of material was loaded onto the Tris-HCl gel. The gel was run with a constant current of 35 mA, 150 W, and the voltage was greater than 700 V for 5.5 h in a cold room, with a water bath set to 12 °C using a Hoefer SE600 electrophoresis unit.
Unphosphorylated and phosphorylated KaiC_RS-Δcoil were separated by Zn²⁺ Phos-tag SDS–PAGE with 10% acrylamide gel containing 50 μM Phos-tag acrylamide (Wako). The gel was run with a constant current of 30 mA for 5 h 30 min in a cold-room, with 1 μg per well protein samples pre-heated at 95 °C for 3 min.
The gels were stained overnight at room temperature with InstantBlue protein gel stain (Expedeon) with gentle shaking and destained with distilled water until bands were clearly visible. The gels were imaged on a ChemiDoc Imager (Bio-Rad), and Image Lab software (Bio-Rad) was used for analysis.

For Western blotting, homogenized protein lysates were diluted using Laemmli buffer, and 20 μg of protein was separated by 12% SDS-PAGE. Proteins were transferred using a semidry blotter. Membranes were blocked in Odyssey blocking buffer in Tris-buffered saline (LI-COR Biosciences, 927-50000) and then incubated with primary antibody for SOD2 (Cell Signaling Technology, 13194) at a dilution of 1:750 in blocking buffer. IRDye 800CW goat anti-rabbit secondary antibody (LI-COR Biosciences, 925-32210) was diluted 1:10,000 in blocking buffer. Antibody signal was detected using the Odyssey CLx imaging system (LI-COR Biosciences), and images were acquired and analyzed using Image Studio acquisition software (LI-COR Biosciences). Total protein staining was carried out using InstantBlue Protein Gel Stain (Expedeon, ISB1L) and then imaged using a ChemiDoc XBS+ molecular imager (Bio-Rad) and Image Lab software (Bio-Rad).