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PNAS 103 (44): 16153-16158

Copyright © 2006 by the National Academy of Sciences.


BIOLOGICAL SCIENCES / BIOCHEMISTRY

Isoelectric focusing technology quantifies protein signaling in 25 cells

Roger A. O'Neill*,, Arunashree Bhamidipati, Xiahui Bi{dagger},, Debabrita Deb-Basu, Linda Cahill, Jason Ferrante, Erik Gentalen, Marc Glazer{ddagger},, John Gossett, Kevin Hacker, Celeste Kirby{dagger},, 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, and Peter B. Vander Horn

Cell Biosciences, Inc., 1050 Page Mill Road, Palo Alto, CA 94304

Communicated by Harvey F. Lodish, Whitehead Institute for Biomedical Research, Cambridge, MA, September 13, 2006

Received for publication August 11, 2006.

Abstract: 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.

Key Words: cell signaling • immunoassay • phosphorylation • Western blot • microfluidic


Freely available online through the PNAS open access option.

{dagger}Present address: Wako Pure Chemical Industries, Ltd., 665 Clyde Avenue, Suite B, Mountain View, CA 94043.

{ddagger}Present address: Alexza Pharmaceuticals, 1020 East Meadow Circle, Palo Alto, CA 94306.

§Present address: PDL BioPharma, Inc., 34801 Campus Drive, Fremont, CA 94555.

Present address: Kalinex, Inc., 5941 Optical Court, San Jose, CA 95138.

Author contributions: R.A.O., L.C., E.G., M.G., R.L., A.R., D.R., D.S., D.V., K.V., T.Y., and P.B.V.H. designed research; A.B., X.B., D.D.-B., J.F., E.G., J.G., K.H., C.K., J.K., R.L., C.M., M.M., T.M., U.N., N.P., A.R., D.R., D.S., D.V., K.V., J.Y., and T.Y. performed research; R.A.O., E.G., D.V., and K.V. analyzed data; and R.A.O. wrote the paper.

Conflict of interest statement: All authors are current or former employees of Cell Biosciences, Inc., and so have rights to purchase stock options. There is currently no public market for the stocks represented by these options.

*To whom correspondence should be addressed. E-mail: roneill{at}cellbiosciences.com

© 2006 by The National Academy of Sciences of the USA


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