Anti aβ42 rabbit monoclonal antibody clone h31l21

For Aβ42 dot blots, 7.8 μl of 5 mg/ml brain homogenates were added to 12.2 μl of freshly made 8.2 M guanidine hydrochloride (GuHCl); 82 mM Tris HCl (pH 8.0) (5 M GuHCl final) and mixed for three days on a nutator. 1μl of this solution, containing 1.95 μg total protein in 5M GuHCl was pipetted in triplicate on a gridded nitrocellulose membrane, dried for one hour at 37°C, washed in TBS-T then water, and stained with Ponceau S. Four identical blots were made and incubated in either 1:4000 anti-Aβ42 rabbit monoclonal antibody (clone H31L21, Invitrogen, #700254), 1:4000 anti-Aβ total mouse monoclonal (3D6, Elan), or 5% milk only (primary delete), followed by HRP-conjugated secondary goat anti-rabbit or horse anti-mouse antibodies (Vector Labs, PI-1000, PI-2000). The blots incubated with anti-Aβ antibody or primary delete were developed together with ECL Plus (Peirce) and imaged simultaneously using a FluorChemR imager (ProteinSimple), then quantified with Alphaview software (ProteinSimple). The secondary and primary antibody delete controls indicated no IgG background. Signal intensities were normalized to Ponceau S staining, triplicates averaged, and statistical analyses were performed as described below.

For Aβ42 dot blots, 5 mg/ml brain homogenates were extracted in 1.56 volumes of freshly made 8.2 M guanidine hydrochloride (GuHCl), 82 mM Tris HCl (pH 8.0) (5 M GuHCl final) over three nights on a nutator. 1 μl of GuHCl extracted sample (1.95 μg total protein) was spotted in triplicate on gridded nitrocellulose membrane, dried one hour at 37°C, washed in TBS-T and then water, and stained with Ponceau S. Four identical blots were made and incubated in either 1:4000 anti-Aβ42 rabbit monoclonal antibody (clone H31L21, Invitrogen, #700254), 1:4000 anti-Aβ total mouse monoclonal (3D6, Elan) or 5% milk only (primary delete), followed by HRP-conjugated secondary antibody (Vector Labs, PI-1000). The duplicate blots incubated with anti-Aβ antibody or primary delete were developed together with ECL Plus (Peirce) and imaged simultaneously using a FluorChemR imager (ProteinSimple), then quantified with Alphaview software (ProteinSimple). The secondary and primary antibody delete controls indicated no IgG background. Signal intensities were normalized to Ponceau S staining, triplicates were averaged, and statistics were performed as described below.