Strategies to increase the reproducibility of protein fibrillization in plate reader assays

TY - JOUR

T1 - Strategies to increase the reproducibility of protein fibrillization in plate reader assays

AU - Giehm, Lise

AU - Otzen, Daniel E

N1 - Copyright 2010 Elsevier Inc. All rights reserved.

PY - 2010/5/15

Y1 - 2010/5/15

N2 - 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.

AB - 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.

KW - Fluorescent Dyes

KW - Humans

KW - Kinetics

KW - Parkinson Disease

KW - Recombinant Proteins

KW - Reproducibility of Results

KW - Sodium Dodecyl Sulfate

KW - Thiazoles

KW - alpha-Synuclein

U2 - 10.1016/j.ab.2010.02.001

DO - 10.1016/j.ab.2010.02.001

M3 - Journal article

C2 - 20149780

SN - 0003-2697

VL - 400

SP - 270

EP - 281

JO - Analytical Biochemistry

JF - Analytical Biochemistry

IS - 2

ER -