Detection of proteinase-resistant protein (PrP) in small brain tissue samples from Creutzfeldt-Jakob disease patients.
We describe a short and a sensitive method to isolate PrP in small samples of brain tissue using a one day procedure. The tissue was homogenized in sarkosyl, cleared by low-speed centrifugation, and then ultracentrifuged. The pellet was suspended in 10 mM Tris-HCl, 10% NaCl, 1% sarkosyl, precipitated by centrifugation and re-suspended in the above solution with proteinase K. After digestion, PrP was spun down, electrophoresed on a 15% SDS-polyacrylamide minigel and then electro-transferred to a nitrocellulose membrane. The blots were processed with rabbit polyclonal antibody against hamster PrP27-30. Four bands of PrP with molecular weights of 28-30 kDa, 24-26 kDa, 19-20 kDa, and 16 kDa were clearly detected by Western blot in two samples obtained by brain biopsy. To test the sensitivity and the specificity of our method we also purified PrP from 20, 50 and 100 mg of cerebral cortical tissues taken from six frozen CJD brains and one Alzheimer's disease brain of our collection. All the CJD samples, but not the Alzheimer's disease one, resulted positive by Western blot. In the smallest sample tested (20 mg), there was at least one band (about 25 kDa) of PrP detectable by Western blot. Thus, this is a valid and efficient method for the diagnosis of CJD in small brain tissue samples.
Laboratory of Virology, Instituto Superiore di Sanità, Rome, Italy.
Y G
YG
Xi
F
F
Cardone
M
M
Pocchiari
D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D001923:Brain Chemistry / Q000502:physiology*; D007562:Creutzfeldt-Jakob Syndrome / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D017354:Point Mutation; D018622:PrP 27-30 Protein / Q000302:isolation & purification / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D018622:PrP 27-30 Protein