Novel approaches to the separation and quantitation of proteins by capillary electrophoresis
Abstract
The goal of this research was to develop new techniques for protein separation and enzymatic assay with high resolution and sensitivity. A simple method has been developed to prepare linear polyacrylamide gel filled capillaries for the separation of proteins. When the concentration of the gel is above 3%, electroosmotic flow is significantly reduced due to the viscosity of the gel. Linear gel filled capillaries can be prepared without modifying the capillaries. The void (bubble) formation problem experienced with crosslinked polyacrylamide was circumvented. When performing gel electrophoresis in the presence of the anionic surfactant, sodium dodecyl sulfate (SDS), proteins can be separated according to their sizes. The migration time is related to the molecular weight of the protein. Gel electrophoresis of proteins under native conditions, without SDS, has also been investigated. The separation of proteins in native gels was based on the electrophoretic mobilities of the protein, which is the same as for free zone electrophoresis. The high viscosity of the gel limits the diffusion and offered limited band-broadening and high resolution. Electrophoretic mediated microanalysis was developed to conduct enzyme assays in the capillary system. The mechanism of this method is to utilize the electrophoretic mobilities of the enzyme and substrate to achieve rapid mixing in the capillary and initiate the reaction. Assay protocol requires a narrow zone of enzyme to be introduced into a capillary which has been filled with appropriate substrate solution. Product is generated while enzyme migrates along the capillary. Assays can be performed in constant and zero potential modes. Alkaline phosphatase (ALP) was used as a model enzyme. The low detection limit of 10$\sp{-12}$ M of ALP was achieved in a polyacrylamide gel filled capillary with two hours incubation time. The assay of two enzymes simultaneously has been performed in a single capillary. A simple electrochemical cell interfaced with capillary electrophoresis system has been used as a detection method for the ALP assay using an alternative electroactive substrate. The results indicate that sensitivity of the detection method, product molecule, and the turn over number of the enzyme reaction determined the ultimately sensitivity of the method.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry
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