(454c) Parallel Analysis of Transcription Factors

Gene expression is regulated by transcription factors (TFs), proteins that bind specific sites of chromosomal DNA to enact the response to intracellular and extracellular stimuli. TFs work alone and in concert with other TFs and other proteins to generate the phenotypes that are observed at the cellular level. Given that there are roughly 3,000 human TFs, the potential network of responses and interactions is considerable. However, the most common means for describing activation of a pathway by a stimulus is the measurement of the activation of a single TF. This limited focus potentially ignores important events that contribute to the cellular response. Thus, the measurement of multiple TFs in parallel would be advantageous.

Several methods to analyze multiple TF s in parallel exist. However, these are limited by the number of TFs that can be measured in one assay, relatively poor sensitivity, and limited flexibility. We have sought to develop a new, scalable, flexible, and sensitive approach to analysis of TF levels. Based on magnetic bead affinity separation, we have devised an approach to measure TF levels, beginning with human TFs, NF-kB and Ap1, in parallel with ~10-fold improved sensitivity over existing approaches. Further results and challenges will be described.