A microtiter plate assay for polyglutamine aggregate extension.
Polyglutamine (polyGln) aggregates are neuropathological markers of expanded CAG repeat disorders, and may also play a critical role in the development of these diseases. We have established a highly sensitive, fast, reproducible, and specific assay capable of monitoring aggregate-dependent deposition 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 simplest form of this assay, polyGln aggregates are made from chemically synthesized peptides and immobilized onto microplate wells. These wells are incubated for different times with low concentrations of a soluble biotinylated polyGln peptide. Europium-streptavidin complexation of the immobilized biotin, followed by time-resolved fluorescence detection of the deposited europium, allows us to calculate the rate (fmol/h) of incorporation of polyGln peptides into polyGln aggregates. This assay will make possible basic studies 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 anti-aggregation inhibitors. It will also be useful for detection and quantitation of aggregation-competent polyGln aggregates in biological materials, which may prove to be of critical importance in understanding the disease mechanism.
Berthelier
V
V
Graduate School of Medicine, University of Tennessee Medical Center, 1925 Alcoa Highway, Knoxville, TN 37920, USA.
Hamilton
J B
JB
Chen
S
S
Wetzel
R
R
D006801:Humans; D007700:Kinetics; D008722:Methods; D019636:Neurodegenerative Diseases / Q000378:metabolism; D010455:Peptides / Q000032:analysis* / Q000138:chemical synthesis / Q000378:metabolism; D015203:Reproducibility of Results
D010455:Peptides; C097188:polyglutamine