A previously undescribed isoelectric focusing technology allows cell signaling to be quantitatively assessed in <25 cells. Highresolution 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

Sodium bisulfite-induced cytosine deamination/PCR (CD-PCR) is currently the most sensitive and robust method to determine the methylation status of all cytosines in a specific DNA sequence. The CDPCR products are directly sequenced with Thermosequenase and capillary electrophoresis; peak areas are then used to determine the mole fraction of methylated cytosines at each site in a single analysis. Here we show that, if the original DNA sample is a mixture of methylated and unmethylated DNA, conventional CDPCR discriminates against the sequence originating from the methylated DNA; CDPCR product does not accurately represent the methylation status of the original DNA sample. While CDPCR bias can lead to serious errors when determining methylation levels, the addition of betaine (N,N,N-trimethylglycine) to the PCR reaction buffer reduces this bias to less than 10%.

We describe a whole-capillary, multicolor laser-induced fluorescence scanner for microfluidic protein analysis systems. Separation of proteins is achieved by isoelectric focusing in a short length of fused-silica capillary after which the resolved proteins are immobilized to the capillary wall using photochemistry. The capillary is then evacuated, and fluorescently labeled antibodies are flowed through the capillary to bind to the immobilized proteins. This technique provides high sensitivity, the ability to spatially resolve and quantify proteins, and provides the opportunity for complete automation. Results obtained by fluorescence detection are compared to those obtained by chemiluminescence while offering enhanced resolution and signal stability.

Although capillary electrophoresis is a powerful sequencing technology, the low heat capacity of a capillary can make difficult the precise control of its temperature, particularly when the capillary is heated to reduce compressions in the separation of DNA sequencing fragments. In this paper, we demonstrate that minute oscillations in the capillary's temperature result in significant degradation in the number of theoretical plates, the resolution between adjacent peaks, and the number of bases of DNA sequence determined from the electrophoresis data. Temperature must be held stable to within 0.1 degrees C to obtain long read lengths. A Monte Carlo simulation demonstrates that this degradation is consistent with laminar flow induced by the periodic thermal expansion and contraction of the separation medium.

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