title: Surface adsorption considerations when working with amyloid fibrils in multiwell plates and Eppendorf tubes.
abstract: The accumulation of cross-β-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 Aβ disaggregation in vitro. Here, we show that Aβ 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 Aβ 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 and Murray et al., Protein Sci 2010;19:836-846 is invalid when used as evidence for a disaggregase activity. Thus, we correct the two prior publications reporting that worm or mammalian cell extracts disaggregate Aβ amyloid fibrils in vitro at 37°C (see Corrigenda in this issue of Protein Science). We apologize for misinterpreting our previous data and for any confounding experimental efforts this may have caused.
authors: Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, 92037; Department of Molecular and Experimental Medicine and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, 92037.
authors: Amber N
authors: AN
authors: Murray
authors: Fernando L
authors: FL
authors: Palhano
authors: Jan
authors: J
authors: Bieschke
authors: Jeffery W
authors: JW
authors: Kelly
mesh_terms: D000327:Adsorption; D000388:Air; D016229:Amyloid beta-Peptides / Q000737:chemistry*; D000818:Animals; D052160:Benzothiazoles; D017173:Caenorhabditis elegans / Q000737:chemistry; D002457:Cell Extracts; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D051379:Mice; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000737:chemistry*; D010969:Plastics; D011489:Protein Denaturation; D011791:Quartz; D013844:Thiazoles / Q000378:metabolism*; D013997:Time Factors; D014867:Water
chemical_list: D016229:Amyloid beta-Peptides; D052160:Benzothiazoles; D002457:Cell Extracts; D010446:Peptide Fragments; D010969:Plastics; D013844:Thiazoles; C078592:amyloid beta-protein (1-40); D014867:Water; D011791:Quartz; C009462:thioflavin T
keywords: Abeta; Alzheimer's disease; amyloid; amyloid adsorption; amyloid disaggregation; fibril; thioflavin T
references: Trends Biochem Sci. 2011 Jun;36(6):338-45
references: 21411326
references: Structure. 2010 Oct 13;18(10):1244-60
references: 20947013
references: J Struct Biol. 2001 Aug;135(2):84-93
references: 11580258
references: Biochem Soc Trans. 1991 Aug;19(3):272S
references: 1723700
references: Nature. 2005 Jun 9;435(7043):773-8
references: 15944695
references: J Mol Biol. 2005 Feb 18;346(2):603-16
references: 15670608
references: Biochemistry. 2010 Dec 28;49(51):10831-41
references: 21050007
references: Nat Biotechnol. 1999 Jul;17(7):676-82
references: 10404161
references: Mol Cell. 2006 Aug 4;23(3):425-38
references: 16885031
references: Annu Rev Biochem. 2006;75:333-66
references: 16756495
references: Protein Sci. 2009 Nov;18(11):2231-41
references: 19701939
references: Biochemistry. 2004 Jun 29;43(25):8107-15
references: 15209506
references: Proc Natl Acad Sci U S A. 2002 Dec 24;99(26):16742-7
references: 12481027
references: Mol Biol Cell. 2011 May 15;22(10):1664-76
references: 21441306
references: Proc Natl Acad Sci U S A. 2010 May 4;107(18):8135-40
references: 20404203
references: Proc Natl Acad Sci U S A. 2006 Oct 24;103(43):15806-11
references: 17038504
references: J Mol Biol. 2008 Dec 31;384(5):1157-73
references: 18851977
references: Neurobiol Aging. 2008 May;29(5):707-15
references: 17222479
references: J Biol Chem. 2007 Aug 3;282(31):22376-86
references: 17545160
references: Protein Sci. 1993 Mar;2(3):404-10
references: 8453378
references: Biochem Cell Biol. 2010 Feb;88(1):1-13
references: 20130674
references: Nat Biotechnol. 2001 Mar;19(3):242-7
references: 11231557
references: Langmuir. 2010 Nov 16;26(22):17225-31
references: 20961051
references: Cell. 2012 Nov 9;151(4):778-793
references: 23141537
references: Proc Natl Acad Sci U S A. 2005 Nov 29;102(48):17342-7
references: 16293696
references: Protein Sci. 2010 Apr;19(4):836-46
references: 20162625
references: Science. 2006 Sep 15;313(5793):1604-10
references: 16902091
references: J Colloid Interface Sci. 2003 Oct 1;266(1):74-81
references: 12957584
references: PLoS One. 2011;6(10):e26319
references: 22022600
references: Pharm Res. 1993 Jul;10(7):954-62
references: 8378257
references: J Biol Chem. 2012 Jan 2;287(1):542-556
references: 22081611
references: Eur J Biochem. 2000 Jan;267(2):566-72
references: 10632727