Date of Graduation

7-2015

Document Type

Thesis

Degree Name

Master of Science in Biomedical Engineering (MSBME)

Degree Level

Graduate

Department

Biomedical Engineering

Advisor

Christa Hestekin

Committee Member

Timothy Muldoon

Second Committee Member

Jeffrey Wolchok

Third Committee Member

Shannon Servoss

Abstract

The accumulation of amyloid-beta (Aβ) oligomers is believed to be the driving force behind Alzheimer’s disease (AD) pathogenesis. Due to the metastable nature of Aβ oligomers, the knowledge of Aβ aggregation and accumulation is not well understood. Here, we use capillary electrophoresis (CE) and photo-induced crosslinking of unmodified proteins (PICUP) to learn about the aggregation of Aβ.

First, we explore the effect of capillary coating on Aβ1-42 protein loss using CE. The dynamic coatings tested were PHEA and 50 kDa, 2000 kDa, 5000 kDa, and 8000 kDa PEO. The covalent coating tested was PVA. The results indicated that 2000 kDa PEO allowed for the best recovery of Aβ1-42 with 87%. This was better than the uncoated capillary recovery of 66%, but less than the optimal 90%. The biggest issue encountered was a protein recovery of greater than 100%, which is theoretically impossible. More procedure development should be done to solve this problem.

Next, the PICUP reaction was optimized for analysis of Aβ1-42. Using a novel approach, the goal was to apply PICUP to capture Aβ1-42 oligomers and correlate bands via SDS-PAGE analysis to peaks via CE analysis. The results showed that reactants needed for successful PICUP application interfered with CE detection of Aβ1-42. We tested alternate reactants and several methods of removing reactants after PICUP application. We learned that dialysis was the most promising method of Aβ1-42 detection via CE after PICUP.