Access Type
Open Access Embargo
Date of Award
January 2022
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Chemistry
First Advisor
Thomas H. Linz
Abstract
Proteins must be in their native states to carry out their intended biological functions. Native protein structure requires that proteins be folded into the correct conformations and possess the necessary post-translational modifications (PTMs). These higher-order structures are critical to ensuring that proteins are biologically active and properly interact with neighboring molecules. However, assessing folding conformations and modified protein variants is challenging, and routine sample validation techniques like polyacrylamide gel electrophoresis (PAGE) are not capable of analyzing higher-order structures. Gradient PAGE gels can determine the molecular weight of proteins over a wide mass range, but protein separation resolutions are poor because of high dispersion and comigration, which limits the sensitivity of native PAGE.In this dissertation, a microfluidic thermal gel electrophoresis (TGE) platform was developed to provide high-sensitivity, high-resolution analyses of native proteins over a wide mass range and to separate conformational variants of native proteins. TGE is a novel separation technique that incorporates a thermal gel with thermally tunable viscosity to control the separation resolution of proteins. The introduction of a temperature gradient into the analysis showed that gel viscosity could be adjusted through a thermal dimension, which affords additional flexibility that cannot be attained in conventional static gels. Protein preconcentration was conducted within the same microfluidic device as the separation for easy automation and enabled the analysis of low-abundance proteins. Furthermore, the TGE platform enabled the user to study protein conformational changes and the formation of protein complexes with minimum sample preparation in a rapid, inexpensive analysis.
Recommended Citation
Peli Thanthri, Shakila Hasanthika, "Microfluidic Thermal Gel Electrophoresis For Native Protein Analysis" (2022). Wayne State University Dissertations. 3782.
https://digitalcommons.wayne.edu/oa_dissertations/3782