Optimized Fixation Protocol for Proteomics

For all experiments, a 4% w/v paraformaldehyde (Sigma‐Aldrich #P6148) solution and a 3% v/v glyoxal (Sigma‐Aldrich #128465) solution were used. Paraformaldehyde was dissolved in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4). The glyoxal solution was prepared according to the following protocol:
For ~4 ml glyoxal solution mix:

2.835 ml ddH₂O

0.789 ml ethanol (absolute, for analysis)

0.313 ml glyoxal (40% stock solution from Sigma‐Aldrich, #128465)

0.03 ml acetic acid

Vortex the solution and bring the pH to 4 or 5 by adding drops of 1 M NaOH until respective pH is reached. Check pH with pH indicator paper. The solution should be kept cool and used within a few days, otherwise glyoxal might precipitate. If the stock solution shows precipitation, glyoxal can be redissolved by heating the solution to ~50°C (see also information provided by Sigma‐Aldrich).
Results obtained with glyoxal at pH 5 are shown in all figures, unless noted otherwise (Appendix Fig S2 shows data obtained from glyoxal pH 4). For several control experiments (as noted in the figure legends), the same amount of ethanol was added to the PFA solution.
The fixatives for the SDS–PAGE experiments (Fig 3A, Appendix Fig S7) were prepared so that the final amount of PFA and glyoxal (mixed with the cytosol samples) were 4% and 3%, respectively. As a control for the SDS–PAGE experiments, 0.2% glutaraldehyde (AppliChem #A3166) was added to a 4% PFA solution, as noted in the respective figure legend.