Czirr E, Cottrell BA, Leuchtenberger S, Kukar T, Ladd TB, Esselmann H, Paul S, Schubenel R, Torpey JW, Pietrzik CU, Golde TE, Wiltfang J, Baumann K, Koo EH, Weggen S. Independent generation of Abeta42 and Abeta38 peptide species by gamma-secretase. J Biol Chem. 2008 Jun 20;283(25):17049-54. PubMed Abstract

REAGENTS/MATERIAL:
Antibodies:
Monoclonal antibody Ab9 against amino acids 1–16 of human Aβ IgG_2a and the carboxyl terminus-specific Aβ antibodies BAP24 (anti-Aβ40), BAP15 (anti-Aβ42), and BAP29 (Narlawar et al., 2007), (Levites et al., 2005).  Biotinylated mouse monoclonal anti-Aβ (6E10) (Signet/Covance) recognizing amino acids 1–17 of human Aβ.   Aβ peptides from 1.25 mg of total protein were immunoprecipitated using 25 μl of Dynabeads (Dynal Biotech, Hamburg, Germany) coated with mouse 1E8 monoclonal antibody directed against the amino terminus (17–24) of Aβ (Bayer-Schering AG).  Immunoprecipitated material or conditioned supernatants were separated on 10% Bicine/Tris gels containing 8 M urea (22), and Aβ peptide species were revealed by Western blotting using antibody 1E8.  Cell supernatants were separated on urea SDS-gels, and Aβ peptides were detected by Western blotting with antibody 1E8 tissue culture supernatants were immunoprecipitated with pan-Aβ antibody Ab9 recognizing amino acids 1–16 of the human Aβ sequence.
We thank Dr. Manfred Brockhaus (F. Hoffmann-La Roche Ltd.) for providing carboxyl terminus-specific Aβ antibodies.