Improved electrophoretic separation and immunoblotting of beta-amyloid (A beta) peptides 1-40, 1-42, and 1-43.
Beta-amyloid peptides (A beta peptides) form the main protein component of the amyloid deposits found in the brains of Alzheimer's disease (AD) patients. Soluble A beta peptides, which are proteolytic fragments of the amyloid-precursor protein (APP) are constitutively secreted by cells expressing APP during normal metabolism [1] and are also present in human plasma and cerebrospinal fluid [2]. Missense mutations in Codon 717 of the APP gene are responsible for a small percentage of inherited AD cases (FAD) and increase the amount of A beta peptides containing additional carboxy terminal amino acids (A beta 1-42, A beta 1-43) [3, 4]. Recent findings indicate that FAD mutations in the presenilin 1 and 2 genes also increase the amount of these longer A beta peptides [5]. A beta 1-42 polymerizes more rapidly in vitro [6] than A beta 1-40 and has been identified as the major component of the brain amyloid deposits [7-9]. We recently developed a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system [10] for the separation of these two peptides. Here we describe a modified version of the original SDS-PAGE procedure, which allows the separation of A beta 1-40, A beta 1-42, and A beta 1-43 for the first time. Detection of the three A beta peptides in the lower ng and pg range is realized by optimized silver staining or immunoblot procedures. These nonradioactive methods may validate results obtained by ELISA procedures used to study the metabolic fate of APP. They may help to define the neurotoxic potential of the longer A beta peptides in relation to their aggregation state.
Wiltfang
J
J
Department of Psychiatry, University of Göttingen, Germany. jwilt-fa@gwdg.de
Smirnov
A
A
Schnierstein
B
B
Kelemen
G
G
Matthies
U
U
Klafki
H W
HW
Staufenbiel
M
M
Hüther
G
G
Rüther
E
E
Kornhuber
J
J
D016229:Amyloid beta-Peptides / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D010446:Peptide Fragments / Q000032:analysis*; D012967:Sodium Dodecyl Sulfate
D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; C078592:amyloid beta-protein (1-40); C075222:amyloid beta-protein (1-42); C098994:amyloid beta-protein (1-43); D012967:Sodium Dodecyl Sulfate