Flow cytometric purification of Alzheimer's disease amyloid plaque core protein using thioflavin T.
Amyloid plaque core protein (APCP) of Alzheimer's disease obtained from brain tissue homogenate is difficult to recover in pure form, primarily because of contaminating lipofuscin (LF) granules. Thioflavin T, a fluorescent dye previously used to stain amyloid, was found to bind to APCP but not to lipofuscin. The latter, however, is autofluorescent. Fluorometric studies showed that at 370 nm excitation APCP has a maximal emission at 418 nm, whereas the autofluorescent LP has a maximal emission at 450 nm. This difference in emission permitted the use of a flow cytometer-sorter (FACS 440) for purification of APCP. APCP particles fluoresced distinctly from LF granules on the log blue fluorescence parameter. The two entities were sorted using forward light scatter versus fluorescence. A collection apparatus was designed and prepared to facilitate the collection of large volumes of sheath fluid and particles and to minimize fragmentation of particles during the collection process. The sorted APCP fraction was 98% pure. This work demonstrates how old dyes can be used to perform new tricks and provide a useful method for separating complex protein.
Palutke
M
M
Department of Pathology, Wayne State University School of Medicine, Detroit, Michigan 48201.
KuKuruga
D
D
Wolfe
D
D
Roher
A
A
D000544:Alzheimer Disease / Q000378:metabolism*; D000682:Amyloid / Q000302:isolation & purification*; D016229:Amyloid beta-Peptides; D052160:Benzothiazoles; D001923:Brain Chemistry; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D008062:Lipofuscin / Q000302:isolation & purification; D008856:Microscopy, Fluorescence; D013050:Spectrometry, Fluorescence; D013844:Thiazoles
D000682:Amyloid; D016229:Amyloid beta-Peptides; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D008062:Lipofuscin; D013844:Thiazoles; C009462:thioflavin T