Presentation Title

Electrophoretically Mediated Microanalysis (EMMA): an Application of Capillary Electrophoresis for Performing Enzyme Assays

Start Date

November 2016

End Date

November 2016

Location

HUB 302-12

Type of Presentation

Poster

Abstract

Capillary electrophoresis (CE) is an analytical technique used to separate ions based on their electrophoretic mobility through an applied voltage. This technique uses a thin fused silica capillary that separates components based on their size and charge, and the solution viscosity. Electrophoretically medicated microananlysis (EMMA) is an application of CE that can be used to perform enzyme assays and to determine substrate concentrations by using Michaelis Menten kinetics collected from experimental data. EMMA is an ideal medium in which to conduct enzyme assays because the capillary can serve as a micro reactor to carry out the enzyme substrate reaction as well as acting as a separation column. In a previous experiment conducted by Fujima and Danielson, lactate dehydrogenase (LDH) was investigated using CE for determination of the concentrations of pyruvate and lactate. One experiment we conducted involved converting pyruvate to lactate coupled with NADH conversion to NAD+. Using a capillary filled with pyruvate and NADH in the running buffer a plug of LDH was injected into the capillary and NAD+ production was monitored at 260nm. A second experiment was performed using lactate and NAD+ in the running buffer and the production of NADH was monitored at a detection wavelength of 340 nm. Good linearity was achieved in lactate concentrations ranging from 0.5 -10 mM.

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Nov 12th, 1:00 PM Nov 12th, 2:00 PM

Electrophoretically Mediated Microanalysis (EMMA): an Application of Capillary Electrophoresis for Performing Enzyme Assays

HUB 302-12

Capillary electrophoresis (CE) is an analytical technique used to separate ions based on their electrophoretic mobility through an applied voltage. This technique uses a thin fused silica capillary that separates components based on their size and charge, and the solution viscosity. Electrophoretically medicated microananlysis (EMMA) is an application of CE that can be used to perform enzyme assays and to determine substrate concentrations by using Michaelis Menten kinetics collected from experimental data. EMMA is an ideal medium in which to conduct enzyme assays because the capillary can serve as a micro reactor to carry out the enzyme substrate reaction as well as acting as a separation column. In a previous experiment conducted by Fujima and Danielson, lactate dehydrogenase (LDH) was investigated using CE for determination of the concentrations of pyruvate and lactate. One experiment we conducted involved converting pyruvate to lactate coupled with NADH conversion to NAD+. Using a capillary filled with pyruvate and NADH in the running buffer a plug of LDH was injected into the capillary and NAD+ production was monitored at 260nm. A second experiment was performed using lactate and NAD+ in the running buffer and the production of NADH was monitored at a detection wavelength of 340 nm. Good linearity was achieved in lactate concentrations ranging from 0.5 -10 mM.