ORGN 447 |
| The development of cell permeable multiuse affinity probes (MAPs) for affinity purification of protein complexes and their subsequent localization in living cells requires a high specificity of labeling in order to obtain the necessary signal-to-noise for real-time imaging. We have therefore synthesized a new fluorescent bis-arsenical probe based on the bisarsencal xanthine fluorophore developed by by Tsien and coworkers (Adams et al., 2002). Incorporation of a carboxylate moeity provides three-orders of magnitude in enhanced specificity, and disrupts nonspecific hydrophobic binding interactions that limit the usefullness of current MAPs. Upon expression of tagged proteins in Shewanella oneidensis, one observes high signal-to-noise with minimal background signal permitting real-time imaging to validate protein-protein interactions in living cells. Following immobilization on an affinity matrix, protein complexes can be specifically isolated in a one-step procedure that is automatable for high-throughput protein complex identification. In conclusion, we have developed a robust methodology for the identification and validation of protein complexes. |
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Bioorganic, Metal-Mediated Reactions, and Molecular Recognition
8:00 PM-10:00 PM, Tuesday, 30 August 2005 Washington DC Convention Center -- Hall A, Poster
Division of Organic Chemistry |