Journal: Scientific Reports
Article Title: Multifaceted anti-amyloidogenic and pro-amyloidogenic effects of C-reactive protein and serum amyloid P component in vitro
Figure Lengend Snippet: Interactions of CRP and SAP with fresh and aggregated Aβ(1-40) and D76N β2-m. ( a–f ) Binding of Aβ(1-40) ( a–c ) and D76N β2-m ( d–f ) to immobilized CRP and SAP. CRP and SAP were immobilized on an ELISA plate and incubated with fresh ( f ) and aggregated (ag) Aβ(1-40) and D76N β2-m in Tris-EDTA ( a,d ), Tris-Ca ( b,e ), or MES-Ca buffer ( c,f ). The data are mean ± SD of three independent incubations. Representative data of three independent experiments are shown. ( g,h ) Crosslinking experiments. At 0 h (fresh mixture, f) and at the beginning of the growth phase (aggregated mixture, ag), the reaction mixture containing Aβ(1-40) or D76N β2-m was spiked with 1:20 CRP and SAP, and incubated for 30 min at 37 °C. After BS 3 was added to the mixture, SDS-PAGE (top) and western blotting analysis (bottom) were performed. In western blotting analysis, bound Aβ(1-40) and D76N β2-m were detected with anti-human Aβ(1-40) and β2-m antibodies, respectively.
Article Snippet: D76N β2-m amyloid fibril formation and ThT assay The reaction mixture (200 μL) that contained 30 μM D76N β2-m, 0–1.5 μM CRP, SAP, or AGP, Tris-EDTA (pH 7.5), Tris-Ca (pH 7.5), MES-Ca (pH 7.0), or MES-EDTA buffer (pH 7.0), and 5 μM ThT was incubated with shaking (800 rpm) at 37 °C in a 96-well plate (237105, Thermo Fisher Scientific, Nunc A/S, Roskilde, Denmark) sealed with sealing film.
Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, SDS Page, Western Blot