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Copyright © 2006 by the National Academy of Sciences.
Isoelectric focusing technology quantifies protein signaling in 25 cells
Roger A. O'Neill*,,
Arunashree Bhamidipati,
Xiahui Bi 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, 2006Received 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.
¶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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Present address: PDL BioPharma, Inc., 34801 Campus Drive, Fremont, CA 94555. Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882
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