Separation and Extraction of Proteins and Compounds Using Nanopores
High throughput methods for detection and quantification of protein therapeutics is needed to further analyze potential biomarkers. Molecular weight determination can also be useful in identification and characterization of protein aggregates and potential biomarkers. Current sized-based separations include sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE), capillary sodium dodecyl sulfate gel electrophoresis (capillary SDS-GE) and size exclusion chromatography (SEC). These methods all have disadvantages when separating larger proteins. Disadvantages include being time consuming, having a protein size limitation, and possessing an uneven pore distribution to give band broadening. To combat these issues and disadvantages, our approach uses a technique we introduced called packed capillary electrophoresis (pCE). This consists of capillaries packed will colloidal silica. Specifically, this dissertation reports packed silica od particle diameter 350 nm as a replacement medium that can reduce time, eliminate heterogeneity and separate arbitrarily large proteins. Parameter optimization for the running solution, separation length, and electric field for pCE was completed using protein standards (11-155 kDa). Using 350 nm gives an effective pore radius 60 nm that is needed separate large-scale proteins (>150 kDa), antibody aggregates and proteins masses beyond 1 MDa. Time and resolution comparisons between SEC, SDS-PAGE, and pCE with 350 bare silica colloidal crystals show that this optimized pCE technique is an efficient way to separate large sized proteins. Chapters 4 to 6 details a research project that explores the binding of estrogen and estrogen like compounds to the estrogen receptor (ER) for possible environmental analyses. To help mediate most biological effects within our bodies, hormone beta estradiol must efficiently bind to the estrogen receptor. Due to antagonistic issues from estrogen like compounds, such as bisphenol A (BPA), the binding of beta estradiol may be disrupted. As a replacement for molecularly imprinted polymers (MIPs) used for environmental analysis, which can be time consuming and have low selectivity, we looked to using an epoxide bead surface to attach an estrogen receptor protein. This complex was used to bind and release estrogen and estrogen like compounds at low concentrations. Using liquid chromatography mass spectrometry (LC-MS), calculated recoveries support the difference in binding affinities of beta estradiol and BPA. The use of the epoxide surface with estrogen receptor showed that we can analyze binding affinities of estrogen and estrogen-like compounds to the estrogen receptor at a smaller scale compared to other commercial techniques.
Wirth, Purdue University.
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