Isoelectric focusing technology quantifies protein signaling in 25 cells

• DOI: 10.1073/pnas.0607973103
• Publication Date: 2006-10-20T01:09:53Z
• Authors: Roger A. O'Neill; Arunashree Bhamid...; Xiahui Bi; Debabrita Deb-Basu; Linda Cahill; Jason Ferrante; Erik Gentalen; Marc Glazer; John Gossett; Kevin Hacker; Celeste Kirby; James Knittle; Robert Loder; Catherine Mastroieni; Michael MacLaren; Thomas Mills; Uyen Nguyen; Nineveh Parker; Audie Rice; David Roach; Daniel Suich; David Voehringer; Karl Voss; Jade Yang; Tom Yang; Peter B. Vander Horn
• Keywords: 0303 health sciences; 03 medical and health sciences; Cell Line, Tumor; Humans; Protein Isoforms; Proteins; Isoelectric Point; Isoelectric Focusing; Phosphorylation; Sensitivity and Specificity; Signal Transduction

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.