Capillary and microchip electrophoresis assays of microbial stress response

Prof. Michelle Kovarik, Associate Professor of Chemistry and Co-Director of the Center for Teaching and Learning at Trinity College, Hartford, CT

Abstract:

While gel electrophoresis is a common tool in molecular biology, free zone capillary electrophoresis has had fewer applications in this area. However, capillary and microchip electrophoresis have advantages for biological measurements, including precise quantitation and single-cell capability. While many bioassays measure fluorescent signals using microscopy or flow cytometry, adding a separation step permits simultaneous measurements of many fluorescent indicators in the same spectral channel, allowing for internal standards and multiplexed measurements. In our lab, we apply this principle to two main areas: kinase activity in response to nutrient deprivation and oxidative stress response. I will present capillary electrophoresis separations of peptide substrate reporters for protein kinase B and their application to cell signaling during social development in the amoeba Dictyostelium discoideum and during mating in the ciliate Tetrahymena thermophila. I will also present capillary electrophoresis and microchip electrophoresis separations of reactive oxygen species indicators. These measurements include data single D. discoideum cells, illustrating the capability of this technology to reveal non-genetic heterogeneity through single-cell assays. Results from both projects demonstrate the power of combining microelectrophoretic separations and existing molecular tools to extend biological research.