Tom Weisan Yang

Isoelectric focusing technology quantifies protein signaling in 25 cells

A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. High-resolution capillary isoelectric focusing allows isoforms and individual phosphorylation forms to be resolved, often to baseline, in a 400-nl capillary. Key to the method is photochemical capture of the resolved protein forms. Once immobilized, the proteins can be probed with specific antibodies flowed through the capillary. Antibodies bound to their targets are detected by chemiluminescence. Because chemiluminescent substrates are flowed through the capillary during detection, localized substrate depletion is overcome, giving excellent linearity of response across several orders of magnitude. By analyzing pan-specific antibody signals from individual resolved forms of a protein, each of these can be quantified, without the problems associated with using multiple antibodies with different binding avidities to detect individual protein forms.

Abstract 1262: Simple western and NanoPro analysis of key EGF signaling cascade proteins

Aberrant expression and signaling in the EGF signaling pathway is a common occurrence in a variety of cancers including breast cancer. Understanding how EGF signaling impacts disease progression is key to the development of novel therapeutics. Detection of important protein targets in cancer samples frequently employs Western blot analysis. In-depth phosphorylation analysis often requires 2D gels which are extremely variable and labor intensive, followed by MS analysis. Each of these methods have their advantages and challenges. This study employs the use of two novel nano-scale capillary-based electrophoresis and immunodetection technologies to characterize and validate subtle changes in key protein targets involved in the regulation of disease progression. The Simon system performs size-based separation of proteins, known as Simple Western assays. Alternatively, NanoPro systems utilize charge-based separation via isoelectric focusing (IEF). Both methods utilize capillary electrophoresis (CE) to perform the separation events. Both Simon and NanoPro instruments provide automated workflow eliminating the need for manual processing of multiple steps traditionally needed to carry out either of these separation and detection processes. Proteins from a single sample preparation were analyzed on two platforms: either in a native conformation using charge-based separation (NanoPro assays) or in a denatured state followed by size-based separation (Simple Western assays). Both methods incorporate immunodetection to monitor both subtle and specific changes to key signaling protein targets. Utilizing both platforms allowed for detailed analysis of post-translational modifications (charge-based) and protein abundance (size-based) within a prepared sample. As a validation of synergy between the platforms, we investigated key members of the EGF signaling cascade in different phosphorylation states. We showed that the same cellular lysate preparation can be used for charge- or size-based separation. Advantages of Simple Western and NanoPro assays over gels (1D and 2D) and traditional Westerns include ease of use, minimal user intervention, automatic analysis and excellent reproducibility which ultimate results in better data quality to characterize key regulatory events involved with cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1262. doi:1538-7445.AM2012-1262