O'Neill et al. report a new highly sensitive method that they call probed isoelectric focusing (PIF) for quantifying phosphorylated proteins based on capillary isoelectric focusing. Cell lysates from as few as 25 cells were subjected to isoelectric focusing in capillary tubes along with standards. The proteins were then covalently attached to photoactivatable groups on the capillary walls and probed with specific antibodies that detected the complete spectrum of phosphorylated and unphosphorylated, or only phosphorylated forms of the proteins of interest. The bound antibodies were then detected with horseradish peroxidase and chemiluminescence. Experiments were performed to detect the relative amounts of phosphorylated forms of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), Akt, and Bcl-2. In the experiments monitoring ERK phosphorylation, PIF allowed single and multiply phosphorylated forms of ERK1 and ERK2 to be detected and quantified as a percentage of the total abundance of each ERK isoform. Western blotting using the same panel of antibodies did not resolve the different phosphorylated individual species. PIF is very sensitive, detecting as little as 2 pg/μl of protein or endogenous ERK from as few as 25 cells. This method may be useful in diagnostic assays or experiments where sample size may be limited.
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, P. B. Vander Horn, Isoelectric focusing technology quantifies protein signaling in 25 cells. Proc. Natl. Acad. Sci. U.S.A. 103, 16153-16158 (2006). [Abstract] [Full Text]