Date of this Version
Truong, B. 2020. Characterization of a Split-Nluc Assay for Monitoring Changes in Alpha-Synuclein Solubility in Living Cells. Undergraduate Honors Thesis. University of Nebraska-Lincoln.
Alpha-synuclein is the primary constituent of Lewy Bodies, protein aggregates associated with Parkinson’s Disease, Dementia with Lewy Bodies, and Multiple Systems Dystrophy. Given the terminal nature of these synucleinopathies, the need for an assay that can report on alpha-synuclein aggregation levels in living systems is critical. Western blotting represents one potential approach to this problem; however, this technique can often be limited by selectivity of available antibodies and experimental reproducibility. Herein, we describe a novel luminescence-based assay capable of detecting alpha-synuclein solubility in bacterial cells. This platform can report on the influence of disease-relevant mutations and small molecules. Antibodies are evaluated for use in Western blotting analysis to complement luminesce assay results. Preliminary data indicate that improved antibodies/Western blotting procedures are needed in order to visualize alpha-synuclein expression in this system. Thus, the luminescence-based assay for alpha-synuclein represents an attractive alternative for monitoring alpha-synuclein solubility and could ultimately be used to study the influence of mutations and tool compounds on alpha-synuclein solubility in living systems.