Detecting Kinase Activities from Single Cell Lysate Using Concentration-Enhanced Mobility Shift Assay
Author(s)Cheow, Lih Feng; Sarkar, Aniruddh; Kolitz, Sarah; Han, Jongyoon; Lauffenburger, Douglas A.
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Electrokinetic preconcentration coupled with mobility shift assays can give rise to very high detection sensitivities. We describe a microfluidic device that utilizes this principle to detect cellular kinase activities by simultaneously concentrating and separating substrate peptides with different phosphorylation states. This platform is capable of reliably measuring kinase activities of single adherent cells cultured in nanoliter volume microwells. We also describe a novel method utilizing spacer peptides that significantly increase separation resolution while maintaining high concentration factors in this device. Thus, multiplexed kinase measurements can be implemented with single cell sensitivity. Multiple kinase activity profiling from single cell lysate could potentially allow us to study heterogeneous activation of signaling pathways that can lead to multiple cell fates.
DepartmentMassachusetts Institute of Technology. Department of Biological Engineering; Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science; Massachusetts Institute of Technology. Research Laboratory of Electronics
American Chemical Society (ACS)
Cheow, Lih Feng, Aniruddh Sarkar, Sarah Kolitz, Douglas Lauffenburger, and Jongyoon Han. “Detecting Kinase Activities from Single Cell Lysate Using Concentration-Enhanced Mobility Shift Assay.” Anal. Chem. 86, no. 15 (August 5, 2014): 7455–7462.
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