Strategies to increase the reproducibility of protein fibrillization in plate reader assays.
There is great interest in developing reproducible high-throughput screens to identify small molecular inhibitors of protein fibrillization and aggregation for possible therapy against deposition diseases such as Alzheimer's and Parkinson's (PD). We have made a methodical analysis of factors increasing the reproducibility of the fibrillization of alpha-synuclein (alphaSN), a 140-amino-acid protein implicated in PD and notorious for its erratic fibrillization behavior. Salts and polyanionic polymers do not significantly improve the quality of the assay. However, an orbital agitation mode in the plate reader is a crucial first step toward reproducible alphaSN fibrillization. Higher reproducibility is achieved by the addition of glass beads, as evaluated by the percentage standard deviation of the nucleation and elongation rate constants and the end-stage fluorescence intensity of the fibril-binding dye thioflavin T (ThT). The highest reproducibility is obtained by either seeding the solution with preformed fibrils or by adding submicellar amounts of sodium dodecyl sulfate (SDS), where we obtain percentage standard deviations of 3-4% on the end ThT level. We conclude that there are multiple ways to achieve satisfactory levels of reproducibility, although the different conditions used to induce aggregation may lead to different fibrillization pathways.
Giehm
Lise
L
Interdisciplinary Nanoscience Center (iNANO), Aarhus University, DK-8000 Aarhus C, Denmark.
Otzen
Daniel E
DE
D052160:Benzothiazoles; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D010300:Parkinson Disease / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry; D051844:alpha-Synuclein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D052160:Benzothiazoles; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D013844:Thiazoles; D051844:alpha-Synuclein; C009462:thioflavin T; D012967:Sodium Dodecyl Sulfate