Burton CR, Meredith JE, Barten DM, Goldstein ME, Krause CM, Kieras CJ, Sisk L, Iben LG, Polson C, Thompson MW, Lin XA, Corsa J, Fiedler T, Pierdomenico M, Cao Y, Roach AH, Cantone JL, Ford MJ, Drexler DM, Olson RE, Yang MG, Bergstrom CP, McElhone KE, Bronson JJ, Macor JE, Blat Y, Grafstrom RH, Stern AM, Seiffert DA, Zaczek R, Albright CF, Toyn JH. The amyloid-beta rise and gamma-secretase inhibitor potency depend on the level of substrate expression. J Biol Chem. 2008 Aug 22;283(34):22992-3003. PubMed Abstract

REAGENTS/MATERIAL:
Aβ-(1-40) was assayed using the mouse monoclonal antibody TSD9S3.2 (Bristol-Myers Squibb), specific for the free C-terminus of Aβ-(1-40), in combination with a peroxidase conjugate of mouse monoclonal anti-Aβ (26D6), specific for an epitope within the 12 N-terminal amino acids of human Aβ-(1-40), or with a peroxidase conjugate of mouse monoclonal anti-Aβ (252Q6) (Biosource/Invitrogen), specific for an epitope within the 12 N-terminal amino acids of rodent Aβ-(1-40). Human Aβ-(1-x) was assayed using monoclonal antibody 26D6, in combination with a biotin conjugate of mouse monoclonal anti-Aβ (4G8) (Signet/Covance), and streptavidin-peroxidase conjugate (Zymed/Invitrogen). This antibody combination detects Aβ peptides containing the 1-24 amino acid region. βCTF levels in cell cultures were determined by quantitative western blotting in comparison to C100 (C99 with additional N-terminal methionine) expressed and purified from Escherichia coli. Gels were blotted onto nitrocellulose membranes and probed with rabbit anti-βAPP (CT695) (Zymed/Invitrogen)