A microtiter plate assay for polyglutamine aggregate extension

Citation
V. Berthelier et al., A microtiter plate assay for polyglutamine aggregate extension, ANALYT BIOC, 295(2), 2001, pp. 227-236
Citations number
43
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ANALYTICAL BIOCHEMISTRY
ISSN journal
00032697 → ACNP
Volume
295
Issue
2
Year of publication
2001
Pages
227 - 236
Database
ISI
SICI code
0003-2697(20010815)295:2<227:AMPAFP>2.0.ZU;2-F
Abstract
Polyglutamine (polyGln) aggregates are neuropathological markers of expande d CAG repeat disorders, and may also play a critical role in the developmen t of these diseases. We have established a highly sensitive, fast, reproduc ible, and specific assay capable of monitoring aggregate-dependent depositi on of polyglutamine peptides. This assay allows detailed studies on various aspects of aggregation kinetics, and also makes possible the detection and quantitation of low levels of "extension-competent" aggregates. In the sim plest form of this assay, polyGln aggregates are made from chemically synth esized peptides and immobilized onto microplate wells. These wells are incu bated for different times with low concentrations of a soluble biotinylated polyGln peptide. Europium-streptavidin complexation of the immobilized bio tin, followed by time-resolved fluorescence detection of the deposited euro pium, allows us to calculate the rate (fmol/h) of incorporation of polyGln peptides into polyGln aggregates. This assay will make possible basic studi es on the assembly mechanism of polyGln aggregates and on critical features of the reaction, such as polyGln length dependence. The assay also will be a valuable tool for screening and characterizing antiaggregation inhibitor s. It will also be useful for detection and quantitation of aggregation-com petent polyGln aggregates in biological materials, which may prove to be of critical importance in understanding the disease mechanism. (C) 2001 Acade mic Press.