Purification and analysis of prion and amyloid aggregates.
Amyloids and prions represent aggregates of misfolded proteins, which consist of protein polymer fibrils with cross-beta sheet structure. Understanding of their occurrence and role is developing rapidly. Initially, they were found associated with mammalian diseases, mainly of neurodegenerative nature. Now they are known to relate to a range of non-disease phenomena in different species from mammals to lower eukaryotes. Uncovering new prion- and amyloid-related processes may be helped greatly by a procedure for purification of amyloid polymers. Studies of growth and propagation of these polymers require methods for determination of their size. Here, we describe such methods. They rely on the treatment with cold SDS or Sarcosyl detergents, which do not dissolve amyloids, but solubilize almost all non-amyloid complexes and associations between amyloid fibers. This allows purifying amyloids by centrifugation in the presence of these detergents. The size of amyloid polymers may be analyzed by electrophoresis in agarose gels containing SDS. Two procedures are described for determining the proportion between polymers and monomers of a particular protein using polyacrylamide gels.
Kushnirov
Vitaly V
VV
Laboratory for Molecular Genetics, Institute of Experimental Cardiology, Cardiology Research Center, 3rd Cherepkovskaya Street 15A, 121552 Moscow, Russia. vkushnirov@cardio.ru
Alexandrov
Ilya M
IM
Mitkevich
Olga V
OV
Shkundina
Irina S
IS
Ter-Avanesyan
Michael D
MD
D000682:Amyloid / Q000032:analysis* / Q000302:isolation & purification*; D002498:Centrifugation; D003902:Detergents / Q000737:chemistry; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D010454:Peptide Termination Factors; D011328:Prions / Q000032:analysis* / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000302:isolation & purification
D000682:Amyloid; D003902:Detergents; D010454:Peptide Termination Factors; D011328:Prions; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins