Uvp chemidoc itts2 imager

Cell lysates of transfected N2A cells were used for western blotting to match with Aβ40 and Aβ42 measurement by ELISA. For analyses of γ-secretase complex subunits and APP fragments, equal protein quantities according to BCA protein concentration were used to prepare samples for western blotting. For quantification of total Aβ in conditioned medium samples that were first analyzed for Aβ40 and Aβ42 content by ELISA, medium volumes normalized to BCA protein content was precipitated with 6 volumes of cold acetone overnight at −20°C. Samples were centrifuged at 15,000 g for 30 minutes and acetone was discarded without disturbing the pellet. Samples were air-dried in the chemical hood and protein pellets were dissolved in 1× gel loading buffer for western blotting analyses. Samples were resolved by electrophoresis in Bolt 4–12% Bis-Tris Plus Gels in Bolt MES SDS running buffer and transferred using iBlot 2 nitrocellulose transfer stacks (Life Technologies). Membranes were blocked and probed with antibodies in 3% non-fat dry milk in PBS / 0.1% Tween −20 buffer overnight at +4°C. Secondary antibody staining was performed for 1 hour at RT and visualized with WesternBright ECL HRP Substrate Kit (Advansta K-12045) and UVP ChemiDoc-It^TS2 Imager (UVP). Images were quantified using ImageJ (NIH).

BV2 cells were lysed in RIPA buffer (Sigma-Aldrich #R0278) supplemented with Protease/Phosphatase Inhibitor Cocktail (Cell Signaling #5872) with one freeze-thaw cycle and 30 min incubation on ice. Lysates were cleared with 30 min centrifugation at 15,000 g. Protein concentration was measured using the BCA kit (Thermo Fisher #PI-23225) and equal quantities were used to prepare the samples for western blotting. Samples were resolved by electrophoresis with Bolt 4–12% Bis-Tris Plus Gels (Invitrogen) in Bolt MES SDS running buffer (Invitrogen, B0002) and transferred using iBlot 2 nitrocellulose transfer stacks (Invitrogen). Membranes were blocked for 1 hour and probed with antibodies 1:1000–4000 in 3% non-fat dry milk in PBS / 0.1% Tween-20 buffer overnight at +4°C. Secondary antibody staining 1:2000 was applied for 1 hour at RT and visualized with WesternBright ECL HRP Substrate Kit (Advansta, K-12045) and UVP ChemiDoc-It^TS2 Imager (UVP). Images were quantified using ImageJ (NIH).