Cdp star chemiluminescent substrate solution

In order to analyse the expression of platelet APP, Western blot analysis was performed. Five µg acidic platelet extracts (neutralized with PBS pH 9.5) were denatured (10 min 70 °C) and loaded onto 4–12 % Bis–Tris polyacrylamide gel (Invitrogen Life Tech, Darmstadt, Germany) and separated by electrophoresis for 60 min at 200 V. Consecutively, samples were electrotransferred to nylon PVDF Immobilon-PSQ membranes (Millipore, Vienna, Austria) at 30 V for 90 min with 20 % methanol transfer buffer (Invitrogen). Protein detection was performed with Western Breeze Chemiluminescent System (Invitrogen). Thus, membranes were blocked for 30 min with blocking solution at RT on the shaker, then incubated with the primary antibody (Anti-Amyloid beta precursor protein antibody [Y188], abcam, Cambridge, UK, ab32136) overnight at 4 °C. Following, blots were washed and incubated with anti-rabbit antibodies for 30 min at RT, again washed and incubated with CDP-Star chemiluminescent substrate solution (Invitrogen). Imaging was performed with a cooled CCD camera SearchLight camera. In order to quantify the protein amounts in each lane and ensure that there has been an even transfer from gel to membrane, a loading control with αMouse IgG was performed.

Western blots were performed as described by us previously [14 (link)]. Briefly, the tissue was dissolved in 200 µL ice-cold PBS with a protease inhibitor cocktail (P8340, Sigma, Hamburg, Germany), homogenized by using an ultrasonic device (Branson sonifier 250, Danburry) and centrifuged at 16,000× g at 4 °C for 10 min. Protein was determined by the Bradford assay. The non-denatured supernatants (25 µg) were loaded onto a 10% Bis-Tris polyacrylamide gel (Invitrogen, Waltham, MA, USA) and were electrophoresed for 25 min at 200 V. Samples were electrotransferred to nylon PVDF (Immobilon-PSQ membranes (Millipore, Burlington, MA, USA) for 90 min at 30 V with 20% methanol blotting buffer (Invitrogen). Blots were blocked for 30 min with blocking buffer, then incubated overnight at 4 °C with the primary serpinin antibody (1:1000; gift from Peng Loh, National Institutes of Health, Bethesda, MD, USA; polyclonal antibody, raised in rabbits) or overnight at 4 °C with a rabbit anti-actin antibody (1:1000, Sigma-Aldrich, St. Louis, MO, USA). Blots were washed and incubated with alkaline phosphatase-conjugated anti-rabbit antibodies for 30 min at room temperature. After washing, blots were incubated (free-floating) in CDP-Star chemiluminescent substrate solution (Invitrogen) for 10 min and the signal was visualized (exposure time 1200 s) with a cooled CCD camera (SearchLight, Thermoscience).