Figure 2

Dot blot assay effectively measures relative levels of cerebral Aβ42. (A) Validation of blot assay. Brain homogenates from 6 month-old female mice were extracted overnight in GuHCl, then spotted in triplicate on nitrocellulose membrane, dried, stained with Ponceau S, and incubated with anti-Aβ42 antibody followed by HRP-conjugated secondary antibody and chemiluminescence imaging. To subtract non-specific background, a duplicate membrane was prepared in the same way, except that the anti-Aβ42 primary antibody was not included (secondary only). (+) or (−) indicate 5XFAD or non-transgenic genotypes, respectively; BACE1 genotype is indicated below 5XFAD genotype. (B) Relative quantification of the Aβ42 dot blot in (A). The secondary only background was subtracted from the Aβ42 immunosignal, then the difference was normalized to Ponceau S staining intensity and triplicates averaged. Group means were calculated and presented as percentages of the 5XFAD/BACE1^+/+ female group. The Aβ42 level in 5XFAD/BACE1^−/− females is not significantly different than that of non-Tg BACE1^+/+ females, indicating that the anti-Aβ42 antibody does not recognize full-length APP. 5XFAD/BACE1^+/− mice have significantly less Aβ42 than 5XFAD/BACE1^+/+ mice and significantly more Aβ42 than 5XFAD/BACE1^−/− mice. n = 6 per genotype, except n = 2 for non-Tg/BACE1^−/−. (C) Aβ42 dot blot demonstrating the dynamic range of the assay. Brain homogenates from female 4, 6, and 9 month-old 5XFAD/BACE1^+/+ (n = 6), 6 month-old non-Tg BACE1^+/+ (n = 6), and BACE1^−/− (n = 2) mice were prepared as in (A), except homogenates were extracted in GuHCl for 72 rather than 24 hrs, which eliminated the IgG background. Since there was no secondary only signal, this blot is not shown, or used for correction. (D) Relative quantification of the Aβ42 dot blot in (C) shows at least a 10-fold dynamic range of detection, and clear differences are observed between groups.