Defined buffer pH gradients for isoelectric focusing

Noah Isaac Tracy

Isoelectric focusing (IEF) requires a pH gradient, which is typically formed using synthetic carrier ampholyte mixtures (SCAMs). Researchers have studied defined buffers as an alternative to SCAMs because they cost less and do not have the adverse interactions with proteins that SCAMs can have. In addition, it has been shown that defined buffers can be used to form shallow pH gradients, which may be useful for separating protein isoforms by IEF. This thesis investigates whether shallow pH gradients formed by defined buffers can be used in IEF to separate protein isoforms. Three defined buffer systems were tested in a vortex-stabilized electrophoresis device to determine which ones would have pH gradients that are useful for separating protein isoforms. The defined buffer pH gradients were formed naturally by applying an electric field or by pouring a concentration gradient of two-components. Only the two-component poured pH gradients were smooth and reproducible. One of the two-component poured pH gradients was used to focus 20 mg of cytochrome c. A second two-component system separated glucose oxidase and amyloglucosidase. These results showed that two-component poured pH gradients can focus and separate proteins. However, the wide amyloglucosidase bands suggest that isoforms may only be partially resolved. A mathematical model to predict the behavior of two-component poured pH gradients during IEF was developed so that computer simulation could be used to test whether or not a particular buffer pair would form a desired gradient for separating proteins. The model used electro-transport theory and equilibrium between the charged states for each component to describe the behavior of the components during IEF. The model was applied to predict pH gradients using the same components as in the first investigation. The model’s predictions fell within the 95% confidence interval of a least-squares fit to the experimental pH gradients except in the regions near the ends of the pH gradients. The model could not predict changes at the ends of the pH gradients because constant composition boundary conditions were assumed. The effects of temperature and concentration on the equilibrium constants, which were neglected for simplicity, may also contribute to the discrepancy between the model and the data.

Defined buffer pH gradients for isoelectric focusing

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