Investigating in vitro kinetics of superoxide dismutase-1 (SOD1) aggregation with high-throughput microplate-based assays provides valuable information regarding SOD1 pathogenesis in amyotrophic lateral sclerosis (ALS) and opens venues for the development of effective therapies. In this chapter, we first explain the step-by-step purification and demetallation of wild-type (WT) and ALS-variant SOD1 proteins from Saccharomyces cerevisiae (baker’s yeast). We then describe the methodology for a microplate-based fluorescence assay that is used to study real-time kinetics of metal-free (apo)-SOD1 aggregation. This technique is highly sensitive, semiautomated, requires minimum modifications to protein, and produces a plethora of data in a short period of time. We also describe a new approach for extracting clinically relevant information from SOD1 aggregation data using Kaplan–Meier estimators.
CITATION STYLE
Abdolvahabi, A., Rasouli, S., Croom, C. M., & Plewman, D. L. (2019). High-throughput microplate-based fluorescence assays for studying stochastic aggregation of superoxide dismutase-1. In Methods in Molecular Biology (Vol. 1873, pp. 93–108). Humana Press Inc. https://doi.org/10.1007/978-1-4939-8820-4_6
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