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  • Title: High-Throughput Microplate-Based Fluorescence Assays for Studying Stochastic Aggregation of Superoxide Dismutase-1.
    Author: Abdolvahabi A, Rasouli S, Croom CM, Plewman DL.
    Journal: Methods Mol Biol; 2019; 1873():93-108. PubMed ID: 30341605.
    Abstract:
    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.
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