Surface adsorption considerations when working with amyloid fibrils in multiwell plates and Eppendorf tubes

Abstract

The accumulation of cross-b-sheet amyloid fibrils is the hallmark of amyloid diseases. Recently, we reported the discovery of amyloid disaggregase activities in extracts from mammalian cells and Caenorhabditis elegans. However, we have discovered a problem with the interpretation of our previous results as Ab disaggregation in vitro. Here, we show that Ab fibrils adsorb to the plastic surface of multiwell plates and Eppendorf tubes. This adsorption is markedly increased in the presence of complex biological mixtures subjected to a denaturing air-water interface. The time-dependent loss of thioflavin T fluorescence that we interpreted previously as disaggregation is due to increased adsorption of Ab amyloid to the surfaces of multiwell plates and Eppendorf tubes in the presence of biological extracts. As the proteins in biological extracts denature over time at the air-water interface due to agitation/shaking, their adsorption increases, in turn promoting adsorption of amyloid fibrils. We delineate important control experiments that quantify the extent of amyloid adsorption to the surface of plastic and quartz containers. Based on the results described in this article, we conclude that our interpretation of the kinetic fibril disaggregation assay data previously reported in Bieschke et al., Protein Sci 2009;18:2231-2241 andMurray et al.,

Human Hsp70 Disaggregase Reverses Parkinson’s-Linked α-Synuclein Amyloid Fibrils

“…The existence of a powerful amyloid disassembly activity by a human chaperone system contrasts with earlier reports showing either no activity (Bieschke et al, 2009; Evans et al, 2006; Murray et al, 2010, 2013) or weak, up to 720-fold lower, activity, requiring 30-day incubations with chaperones for 50% disassembly (Duennwald et al, 2012). Failure to detect robust Hsp70-based disassembly activity in earlier studies probably reflects the specificity of the Hsp70 machine assembled.…”

“…Metazoans, however, entirely lack Hsp100 disaggregases in the cytosol and nucleus, and efficient in vitro disassembly of amyloid aggregates on a physiologically relevant timescale (minutes to hours), has so far not been demonstrated (Bieschke et al, 2009; Duennwald et al, 2012; Evans et al, 2006; Murray et al, 2010, 2013). Metazoans do nonetheless solubilize amorphous, stress-induced protein aggregates, both in vivo and in vitro (Rampelt et al, 2012; Shorter, 2011).…”

The Kinetic Stability of a Full-Length Antibody Light Chain Dimer Determines whether Endoproteolysis Can Release Amyloidogenic Variable Domains

“…3b). The decrease in ThT fluorescence of the V-domain amyloid fibrils over the duration of the experiment is likely due to large aggregates adhering to the wells [38], increased light scattering, and/or structural annealing that alters ThT binding as the fibrils age [37]. Both V-domains aggregated similarly in PBS when monitored by ThT fluorescence in a cuvette at 37 °C with vigorous stirring, with kinetics characteristic of a nucleated polymerization (Fig.…”

Facile measurement of protein stability and folding kinetics using a nano differential scanning fluorimeter

“…In such cases, fluorescently labeled proteins can be used for improved sensitivity. However, labeling can affect the stability of the protein or produce experimental anomalies . Further use of plastic microtiter plates can lead to sample adsorption issues that may lead to incorrect data interpretations, and variations in the meniscus position between wells can also affect the measurement.…”