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Isoelectric focusing technology quantifies protein signaling in 25 cells

PNAS, 31 October 2006
Vol. 103, Issue 44, p. 16153-16158
DOI: 10.1073/pnas.0607973103

Isoelectric focusing technology quantifies protein signaling in 25 cells

  1. Roger A. O'Neill *,
  2. Arunashree Bhamidipati,
  3. Xiahui Bi ,
  4. Debabrita Deb-Basu,
  5. Linda Cahill,
  6. Jason Ferrante,
  7. Erik Gentalen,
  8. Marc Glazer ,
  9. John Gossett,
  10. Kevin Hacker,
  11. Celeste Kirby ,
  12. James Knittle,
  13. Robert Loder,
  14. Catherine Mastroieni,
  15. Michael MacLaren,
  16. Thomas Mills,
  17. Uyen Nguyen,
  18. Nineveh Parker,
  19. Audie Rice § ,
  20. David Roach,
  21. Daniel Suich ,
  22. David Voehringer,
  23. Karl Voss,
  24. Jade Yang,
  25. Tom Yang, and
  26. Peter B. Vander Horn
  1. Cell Biosciences, Inc., 1050 Page Mill Road, Palo Alto, CA 94304
  1. Communicated by Harvey F. Lodish, Whitehead Institute for Biomedical Research, Cambridge, MA, September 13, 2006 (received for review 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.

  • cell signaling
  • immunoassay
  • phosphorylation
  • Western blot
  • microfluidic

Footnotes

  • *To whom correspondence should be addressed. E-mail: roneill{at}cellbiosciences.com
  • Present address: Wako Pure Chemical Industries, Ltd., 665 Clyde Avenue, Suite B, Mountain View, CA 94043.

  • 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.

  • Abbreviations:
    IEF,
    isoelectric focusing;
    PIF,
    probed isoelectric focusing
  • Freely available online through the PNAS open access option.

Citation:

R. A. O'Neill, A. Bhamidipati, X. Bi, D. Deb-Basu, L. Cahill, J. Ferrante, E. Gentalen, M. Glazer, J. Gossett, K. Hacker, C. Kirby, J. Knittle, R. Loder, C. Mastroieni, M. MacLaren, T. Mills, U. Nguyen, N. Parker, A. Rice, D. Roach, D. Suich, D. Voehringer, K. Voss, J. Yang, T. Yang, and P. B. Vander Horn, Isoelectric focusing technology quantifies protein signaling in 25 cells. PNAS 103, 16153-16158 (2006).

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