The 2DE is a powerful proteomic technique, with excellent protein separation capabilities where intact proteins are spatially separated by pI and molecular weight. 2DE is commonly used in conjunction with MS to identify proteins of interest. Current 2DE workflow requires several manual processing steps that can lead to experimental variability and sample loss. One such step is the transition between first dimension IEF and second-dimension SDS-PAGE, which requires exchanging denaturants and the reduction and alkylation of proteins. This in-solution-based equilibration step has been shown to be rather inefficient, losing up to 30% of the original starting material through diffusion effects. We have developed a refinement of this equilibration step using agarose stacking gels poured on top of the second-dimension SDS-PAGE gel, referred to as in-gel equilibration. We show that in-gel equilibration is effective at reduction and alkylation in SDS-PAGE gels. Quantification of whole-cell extracts separated on 2DE gels shows that in-gel equilibration increases protein retention, decreased intergel variability, and simplifies 2DE workflow.

Wnt signaling generates patterns in all embryos, from flies to humans, and controls cell fate, proliferation and metabolic homeostasis. Inappropriate Wnt pathway activation results in diseases, including colorectal cancer. The adenomatous polyposis coli (APC) tumor suppressor gene encodes a multifunctional protein that is an essential regulator of Wnt signaling and cytoskeletal organization. Although progress has been made in defining the role of APC in a normal cellular context, there are still significant gaps in our understanding of APC-dependent cellular function and dysfunction. We expanded the APC-associated protein network using a combination of genetics and a proteomic technique called twodimensional difference gel electrophoresis (2D-DIGE). We show that loss of Drosophila Apc2 causes protein isoform changes reflecting misregulation of post-translational modifications (PTMs), which are not dependent on β-catenin transcriptional activity. Mass spectrometry revealed that proteins involved in metabolic and biosynthetic pathways, protein synthesis and degradation, and cell signaling are affected by Apc2 loss. We demonstrate that changes in phosphorylation partially account for the altered PTMs in APC mutants, suggesting that APC mutants affect other types of PTM. Finally, through this approach Aminopeptidase P was identified as a new regulator of β-catenin abundance in Drosophila embryos. This study provides new perspectives on the cellular effects of APC that might lead to a deeper understanding of its role in development.