High-Throughput Electrophoretic Assays of Proteins, Protein-Protein Interactions, and Protein Function — ASN Events

High-Throughput Electrophoretic Assays of Proteins, Protein-Protein Interactions, and Protein Function (#3)

Robert Kennedy 1
  1. University of Michigan, Ann Arbor, MI, United States

Capillary and microchip electrophoresis (CE or MCE) are well-suited for protein analysis because of the potential for high efficiency and short analysis times based on the use of high electric fields and low diffusion coefficients of proteins. We have explored several novel ways of studying proteins by CE and MCE. The most common protein assay is Western blot, but this method is slow and manually intensive. We have developed a microchip Western that takes advantage of the high speed separation of proteins. In this approach, separated proteins exiting a chip are captured on a membrane for immunoanalysis enabling several Western assays to be performed in 25 min (< 5min/assay). We have also developed an approach to detect protein-protein interactions (PPI) using CE. In the method, one protein is labeled and allowed to bind with a partner in solution. The resulting mixture is separated by CE. Because separation faster than dissociation, the complex can be detected and quantified. We describe an interaction of Hsp70 with modulating proteins assayed by CE. Protein function can readily measured by using CE or MCE as the "readout" for an enzyme assay. We have developed several CE enzyme assays for sirtuins and HDACs enzymes that have significant advantages over existing fluorescent assays based on the added information provided by separation. Because PPI and enzymes are important drug targets, these assays have the potential to be used as high-throughput screens for drug discovery. This requires developing methods to rapidly inject and separate different samples onto chips. We have developed a segmented flow interface to MCE that allows samples stored as nanoliter droplets to be introduced at rates up to 1 Hz onto a chip. These methods are being explored for important new drug targets.