Table of Contents
International Journal of Proteomics
Volume 2012 (2012), Article ID 473843, 7 pages
http://dx.doi.org/10.1155/2012/473843
Research Article

Rapid Screening of the Epidermal Growth Factor Receptor Phosphosignaling Pathway via Microplate-Based Dot Blot Assays

EMD Millipore, Merck KGaA, 17 Cherry Hill Drive, Danvers, MA 01923, USA

Received 4 June 2012; Accepted 15 July 2012

Academic Editor: Gary B. Smejkal

Copyright © 2012 Amedeo Cappione et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Expression profiling on a large scale, as is the case in drug discovery, is often accomplished through use of sophisticated solid-phase protein microarrays or multiplex bead technologies. While offering both high-throughput and high-content analysis, these platforms are often too cost prohibitive or technically challenging for many research settings. Capitalizing on the favorable attributes of the standard ELISA and slot blotting techniques, we developed a modified dot blot assay that provides a simple cost-effective alternative for semiquantitative expression analysis of multiple proteins across multiple samples. Similar in protocol to an ELISA, but based in a membrane bound 96-well microplate, the assay takes advantage of vacuum filtration to expedite the tedious process of washing in between binding steps. We report on the optimization of the assay and demonstrate its use in profiling temporal changes in phosphorylation events in the well-characterized EGF-induced signaling cascade of A431 cells.