Two-Dimensional Gel Electrophoresis of Proteins

2D gel electrophoresis was performed as described by Jameson-Lee et al.⁴¹ (link) Briefly, eluted samples were boiled with SDS-PAGE loading buffer and resolved on Bio-Rad Criterion Precast gel (12 well, 4–12% acrylamide) under nonreducing conditions. The lanes were excised and treated in warm 100 mM DTT for 15 min. Free sulfhydryls were alkylated by 100 mM iodoacetamide in Laemmli loading buffer without bromophenol blue for 5 min. The gel strip was then placed in a 1-well Criterion Precast gel (6–16% acrylamide). The strip was locked in place by addition of an agarose overlay (2-D starter Kit, Bio-Rad) before gel electrophoresis. Proteins were visualized with silver stain.