Xcell surelock electrophoresis cell

Folding of denatured protein was initiated by diluting the crude protein fractions 1/20 (v/v) in detergent buffer (1% (w/v) sulfobetaine 12, 50 mM Tris-HCl, pH 8.0). The folding was performed at 15 °C on a PCMT Thermo-Shaker at 1000rpm for up to 64 min 10uL of the sample was quenched in 10uL of ice-cold SDS-PAGE sample buffer (8% SDS, 40% Glycerol, 50 mM Tris/HCl pH 6.8, 2 mM EDTA, 0.1% bromophenol blue) at specific time points. Three technical replicates were produced for each data point of the folding kinetics experiments.
Samples were kept at 4°C and applied to a pre-cast Novex WedgeWell 4–20% Tris-Glycine polyacrylamide gel (Invitrogen) along with an unfolded control and ladder (PageRuler™ Plus Prestained Protein Ladder, 10–250 kDa). A VWR 250V power source and a xCell SureLock™ Electrophoresis Cell (Invitrogen) was used for electrophoresis at a constant 225V in Tris-glycine buffer. The electrophoresis chamber was kept on ice for the duration of the electrophoresis. Ensuring consistent voltage, current and temperature across all sampled gels was essential to measure reproducible folding kinetics. The gels were stained in 0.125% Coomassie Brilliant Blue R250 (SIGMA) in 50% Ethanol, 10% Acetic acid overnight, and destained in distilled water.

Proteins were resolved using a XCell SureLock™ Electrophoresis cell (Invitrogen, Waltham, MA, USA) on 4–12% NuPAGE Bis-Tris gel (Invitrogen) with MOPS SDS running buffer (Invitrogen) at constant 150 V for 1–1.5 h. The SDS-PAGE gel was stained either with Coomassie blue or silver staining procedures.
For Coomassie blue staining, the gel was first fixed in fixing solution (50% methanol and 10% glacial acetic acid) for 1 h with gentle agitation. Thereafter, the gel was stained for 1–2 h with staining solution containing 0.1% Coomassie Brilliant Blue R-250, 50% methanol and 10% glacial acetic acid. Destaining was done by washing the gel in a solution containing 20% methanol and 5% glacial acetic acid. After staining, images were taken using BioRad GelDoc™ (Bio-Rad Laboratories, Copenhagen, Denmark) or Epson perfection V500 photo scanner (Epson, Herlev, Denmark).
The protein gels were blotted on polyvinylidene fluoride (PVDF) membrane, and the zinc binding protein were visualized by dithizone staining, according to Uddin and co-workers [49 (link)].

For separation of protein by molecular weight in liquid phase recovery GELFREE^® 8100 fractionation system (Expedeon/Protein Discovery Inc., San Diego, CA, USA) was used according to the manufacturer’s protocol. In short, 150 μL protein sample was mixed with 40 μL 5× loading buffer and 10 μL 1 M DTT, and heated for 100 °C for 10 min. Then, 200 μL of sample was loaded into the chamber for separation using the 10% Tris-acetate cartridge (Expedeon Expedeon/Protein Discovery Inc., San Diego, CA, USA) with the mass range 3.5–100 kDa, and resolution between 15 and 100 kDa. The samples were loaded in different loading chambers, and run in different channels so that there was no carryover or cross contamination. Samples were collected from the electroelution chamber, 180 μL each, at established time points over 2–3 h (see Supplementary Figure S1 for details on time points). SDS-PAGE was carried out in XCell SureLock™ Electrophoresis cell (Invitrogen by Thermo Fisher Scientific, Roskilde, Denmark) on 4–12% NuPAGE Bis-Tris gel with MOPS SDS running buffer at constant 120 V for 1–1.5 h. Afterwards, SDS-PAGE gels were fixed in 50% methanol and 1% acetic acid, and stained by silver staining using the PageSilver™ Silver Staining Kit (Fermentas by Thermo Fisher Scientific, Roskilde, Denmark), according to the manufacturer’s procedure.