- 9:50 AM
580e

“Detectorless” Electrophoresis for Multiplexed Enzyme Activity Assays

David Ross, National Institute of Standards and Technology, 100 Bureau Dr., MS 8313, Gaithersburg, MD 20899 and Jason G. Kralj, Biochemical Sciences, National Institute of Standards and Technology, 100 Bureau Dr., MS 8313, Gaithersburg, MD 20899.

A high-throughput separation system was developed by multiplexing gradient elution moving boundary electrophoresis (GEMBE), with each channel acting as separator and conductivity detector. GEMBE functions by combining electrophoresis with a pressure-driven bulk counterflow. The counterflow is gradually reduced until each analyte enters the separation channel and is detected. Each analyte appears as a step in the electrical current vs. time, which is smoothed and differentiated to produce electropherogram peaks. All measurements are electrical rather than optical, making the system easy to scale up to perform large numbers of simultaneous assays in parallel.

Using our prototype system, we demonstrated 16 parallel kinase enzyme activity assays with and without inhibitor; each sampled 13 times over 6 h to observe reaction progress. The other main advantages of this approach are that it excludes proteins and enzymes, eliminating the need for surface coatings or additives to reduce channel fouling; and no stop reagents are needed because the samples are analyzed in real time and parallel.