Determination of NAD+ and NADH in a Single Cell under Hydrogen Peroxide Stress by Capillary Electrophoresis (CAT#: STEM-ET-0296-ZJF)

Capillary Electrophoresis (CE) is physical method of analysis which performs in a separation channel of elastic quartz capillary, under the influence of a high voltage direct current field. Charged analytes dissolved in an electrolyte solution are separated based on differences in mobility and/or distribution behavior of components. The migration velocity of an analyte under an electric field is determined by the electrophoretic mobility of the analyte and the electro-osmotic mobility of the buffer inside the capillary. The electrophoretic mobility of a solute depends on the characteristics of the solute (electric charge, molecular size and shape) and those of the buffer in which the migration takes place (type and ionic strength of the electrolyte, pH, viscosity and additives). Capillary electrophoresis provides greater resolution, higher sensitivity and online detection. It enables single-cell analysis and even single-molecule analysis, optimizing separation and analysis of biological macromolecules.

Biomedical, clinical, pharmaceutical, forensic, industrial, and food analysis

1. Preparation: Preheat the capillary electrophoresis apparatus and flush the capillary with no voltage applied. Prepare mixed standard samples and buffer solution.
2. Sample Application: Put the appropriate amount of mixed standard samples in the sample tube at the corresponding position at the inlet of the capillary electrophoresis apparatus, and put the appropriate amount of buffer in the sample tube at the corresponding position of the apparatus.
3. Electrophoresis: Switch on the electrophoresis apparatus and set the voltage and program. Initiate automatic sampling, electrophoresis, and analysis.
4. Determination: Record and analyze the migration of each component. Capillary electrophoresis apparatus can be connected with various detectors to detect the separation. The most widely used is the UV-visible spectrophotometric detector. After the experiment, flush the capillary again.

• Capillary electrophoresis apparatus
• Sample solution
• Buffer solution

1. When an electric field is applied through the capillary filled with buffer, a flow of solvent is generated inside the capillary, called electro-osmotic flow (EOF). The reproducibility of CE separation will be seriously affected by small changes in EOF. For some applications, it is important to control EOF by modifying the inner wall of the capillary or by changing the concentration, composition and/or pH of the buffer solution.
2. The definition and automation of the injection process are critical for precise quantitative analysis. Modes of injection include gravity, pressure or vacuum injection and electrokinetic injection.
3. The employed electrolytic solution should be filtered to remove particles and degassed to avoid bubble formation that could interfere with the detection system or interrupt the electrical contact in the capillary during the separation run.
4. A rigorous rinsing procedure should be developed to achieve reproducible migration times of the solutes.