Quantikine elisa human amyloid β aa1 40 aa1 42 immunoassay kits

Soluble and insoluble ingredients of Aβ were detected as previously reported (10 (link), 72 (link)). The frozen tissues were homogenized in 15 volumes (w/v) of TBS homogenization buffer, which contained phosphatase and protease inhibitor cocktails (Thermo Fisher Scientific), and centrifuged at 100,000g for 1 hour at 4°C. The supernatant fraction was collected as TBS-soluble fraction, and the sediment fraction was resuspended in 15 volumes (w/v) of 1% Triton X-100/TBS (TBS-X). And then the samples were incubated on ice for 30 minutes followed by centrifuging at 100,000g for 1 hour at 4°C. The supernatant fraction was collected as TBS-X–soluble fraction, and the sediment fraction was resuspended with 15 volumes (w/v) of 70% FA. After the samples were centrifuged at 100,000g for 1 hour at 4°C, the supernatant fraction was neutralized by 20 volumes 1 M Tris base (pH 11), which was named FA-soluble fraction. The protein concentration of TBS-soluble fraction and TBS-X–soluble fraction was measured by BCA protein assay kit (Thermo Fisher Scientific). And protein concentration of FA-soluble fraction was measured by a Bradford protein assay kit (Beyotime). The Aβ₄₀ and Aβ₄₂ levels were quantified with Quantikine ELISA Human Amyloid β aa1-40/aa1-42 immunoassay kits (R&D Systems) according to the manufacturer’s protocol.

Soluble and insoluble Aβ in the brains were sequentially extracted as described elsewhere (Youmans et al., 2011). In brief, brains were homogenized in 15 volumes (w/v) of TBS buffer with phosphatase and protease inhibitor cocktails (Sigma) and then centrifuged at 100 000 g for 1 h at 4°C. The supernatant was collected as the TBS‐soluble fraction (soluble Aβ). The pellet was resuspended in 15 volumes of 1% Triton X‐100/TBS (TBS‐X), incubated on ice for 30 min and then centrifuged at 100 000 g for 1 h at 4°C. The supernatant was removed as the TBS‐X‐soluble fraction. The pellet was resuspended in 70% formic acid (FA) and centrifuged at 100 000 g for 1 h at 4°C. The supernatant was neutralized with 1 M Tris base (pH 11), representing the FA‐soluble fraction (insoluble Aβ). The protein levels of the TBS‐soluble and TBS‐X‐soluble fractions were quantified using a BCA protein assay kit (Bioworlde), and the protein levels of the FA‐soluble fractions were detected using a Bradford protein assay kit (Bioworlde). The levels of Aβ_1–40 and Aβ_1–42 in each fraction were quantified using Quantikine ELISA Human Amyloid β aa1‐40/aa1‐42 Immunoassay kits (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions.