Various concentrations (0. 1 – 100 μM) of compounds were plated in white 384-well plates (Greiner) and were incubated with 10 nM biotinylated Aβ42 (Anaspec) and 1 nM fibrinogen for 30 min at room temperature (RT) in a final volume of 10 μl assay buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20, and 0.1% BSA). The mixture was then incubated with anti-fibrinogen antibody (Dako), 20 μg/ml streptavidin-conjugated donor, and protein A-conjugated acceptor beads (PerkinElmer) for 90 min at RT. Samples were read by a PerkinElmer EnVision plate reader. RU-505, which is a known inhibitor of Aβ42-fibrinogen interaction27 (link), was used as a positive control and also for comparison of its efficacy with other compounds. All the experiments were performed in triplicate. To determine IC50 of compounds, the data were fitted to a sigmoidal dose-response equation (Y= Bottom + (Top − Bottom)/1 + 10(logIC50 − x) Hill coefficient)) using GraphPad Prism 4 to calculate IC50.
Biotinylated aβ42
Manufactured by AnaSpec
Sourced in United States
Biotinylated Aβ42 is a synthetic peptide that consists of the amyloid-beta (Aβ) 42 amino acid sequence with a biotin label attached. This product is designed for use in research applications that require the detection or immobilization of the Aβ42 peptide.
Automatically generated - may contain errors
Lab products found in correlation
Anti fibrinogen antibody, by Agilent Technologies (2 mentions)
White 384 well plate, by Greiner (1 mentions)
Protein a conjugated acceptor beads, by PerkinElmer (1 mentions)
Prism 4, by GraphPad (1 mentions)
Fibrinogen, by Merck Group (1 mentions)
Streptavidin sepharose high performance bead, by GE Healthcare (1 mentions)
Anti aβ antibody 4g8, by Fortrea (1 mentions)
3 protocols using biotinylated aβ42
1
Aβ42-Fibrinogen Interaction Inhibition Assay
2
Investigating Aβ42 and Fibrinogen Interaction
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Hit compounds were tested using a pull-down assay as described previously (Ahn et al., 2010 (link)). In brief, compounds at 10 µM were incubated with 100 nM biotinylated Aβ42 (Anaspec) and 5 nM fibrinogen (EMD Millipore) for 1 h at room temperature in 500 µl of binding buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Nonidet P-40, 0.1% BSA, and protease inhibitor mixture). The samples were gently rotated for 1 h at room temperature with 30 µl streptavidin–Sepharose high performance beads (GE Healthcare). After incubation, the beads were washed five times with binding buffer, and nonreducing sample buffer was added to the beads for elution. Western blots were performed using antifibrinogen antibody (Dako). Dot blots were performed using anti-Aβ antibody 4G8 (Covance) to show comparable amounts of Aβ were also being pulled down.
3
Aβ₄₂ Binding Assay of RI-Peptide Tetramer
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We tested Aβ42 binding activity of the tetramer RI-peptide by ELISA. Microplate wells were coated with 100 ng of the tetramer RI-peptide in carbonate buffer pH 9.6 for 1 h at 37°C. Wells were blocked with 3% gelatin in Tris-buffered saline (TBS) for 1 h at 37°C. To the bound RI-peptide different concentrations (1, 5, and 10 ng) of biotinylated Aβ42 (AnaSpec, CA, USA) was added in triplicate and incubated for 1 h at 37°C. Plates were washed 3 times with TBST and incubated with streptavidin-HRP (1 : 5000) (KPL, Gaithersburg, MD) for 1 h at 37°C. After washing the wells 3 times with TBST, 100 μl HRP substrate solution (SureBlue, KPL, Gaithersburg, MD, USA) was added and incubated for 1 min at RT. One hundred μl of 1 N HCl was added to stop the reaction and the absorbance was read at 450 nm.
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