Purification of recombinant tau protein and preparation of Alzheimer-paired helical filaments in vitro.
The tau protein is a neuronal microtubule-associated protein. Apart of its physiological function-the binding to and stabilization of microtubules-tau is found in Alzheimer's disease brain as insoluble fibers, the so-called "paired helical filaments" (PHFs). Investigating the fundamentals of tau polymerization is indispensable for identifying inhibitory conditions or compounds preventing PHF formation, which may slow down or even stop the degeneration of neurons in Alzheimer's disease. In this chapter, we describe the methods necessary for studying the characteristics of tau polymerization to PHFs. These include: a purification protocol for recombinantly expressed tau; a general method for the polyanion induced polymerization of tau to PHFs; the quantitation of PHFs by a fluorescence-based assay; the imaging and verification of PHFs by negative stain transmission electron microscopy.
Max-Planck-Unit for Structural Molecular Biology, Hamburg, Germany.
Stefan
S
Barghorn
Jacek
J
Biernat
Eckhard
E
Mandelkow
D000544:Alzheimer Disease / Q000378:metabolism*; D000682:Amyloid / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D052160:Benzothiazoles; D003001:Cloning, Molecular / Q000379:methods; D006801:Humans; D008854:Microscopy, Electron / Q000379:methods; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence / Q000379:methods; D013844:Thiazoles; D016875:tau Proteins / Q000235:genetics / Q000302:isolation & purification*
D000682:Amyloid; D052160:Benzothiazoles; D020033:Protein Isoforms; D013844:Thiazoles; D016875:tau Proteins; C009462:thioflavin T