For protein separation, 30 μl of concentrated eluate was fractionated on a NuPAGE 4–12% Bis-Tris Protein gel (ThermoFisher). The gel was fixed overnight in 40% ethanol and 10% acetic acid. The gel was incubated in Sensitizing Solution (30% ethanol, 0.2% sodium thiosulphate and 6.8% sodium acetate) for 30 minutes before being washed three times with water for 5 min each wash. The gel was then stained in 0.25% silver nitrate for 20 min and washed twice more with water for 1 min each time. The bands were visualized by developing in 2.5% sodium carbonate and 0.015% formaldehyde and allowed to incubate until bands appeared. The remainder of each eluate (~65 μl) was loaded on a NuPAGE 4–12% Bis-Tris protein gel and briefly fractionated to yield a single gel band corresponding to the majority of proteins within each purification. Gel bands were excised using a razor blade and subject to in gel reduction, alkylation and trypsin digest by the URMC Mass Spectrometry Resource Lab67,68.
