David J. Roach

Isoelectric focusing technology quantifies protein signaling in 25 cells

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.

Parallel DNA Sequencing on Microfabricated Electrophoresis Chips

We report the fabrication and applications of a 16-channel capillary array electrophoresis (CAE) chip for parallel DNA sequencing. Samples are automatically loaded into sample reservoirs using an 8-tip pipetting device. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated four-color confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels In the best case using an automated base-calling program, 543 bases have been called at an accuracy of>99% and 608 bases at 98%. Separations, including automated chip loading and sample injection, are normally completed in less than 18 min.