Aβ and AICD generated in γ-secretase in vitro assays with purified substrates were analyzed as previously described (28 (link)). Briefly, Aβ peptides were immunoprecipitated overnight using anti-Aβ antibody 4G8 (Covance) and protein G-coupled to agarose resin (Roche Applied Science). For AICD detection, 1% Triton X-100 solution was added after the enzymatic reaction and incubated for 20 min at 55 °C on a shaker prior to overnight immunoprecipitation at 4 °C with protein A (Roche Applied Science), coupled with anti-C-terminal AICD antibody. Aβs and AICDs were eluted with 1:20:20 (v/v/v) 1% (v/v) trifluroacetic acid:acetonitrile:H2O, equally mixed with saturated α-cyano 4-hydroxy cinnaminic acid for detection of Aβs and synapic acid for AICD, and analyzed by MALDI-TOF mass spectrometry in reflectron mode on an ABI 4800 MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Carlsbad, CA). Molecular masses were accurately measured and searched against amino acid sequences of human APP-C99 with the addition of a methionine residue at the N terminus and a His6 tag sequence at the C terminus (C100His). These experiments for each species were performed in duplicate with comparable observed peak height values (Fig. 4 and supplemental Fig. S8b ).
Anti aβ antibody 4g8
Manufactured by Fortrea
The Anti-Aβ antibody 4G8 is a monoclonal antibody that recognizes the amyloid-beta (Aβ) protein. It binds to a specific epitope within the Aβ sequence, allowing researchers to detect and study the Aβ protein in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Abi 4800 maldi tof tof mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Fibrinogen, by Merck Group (1 mentions)
Streptavidin sepharose high performance bead, by GE Healthcare (1 mentions)
Anti fibrinogen antibody, by Agilent Technologies (1 mentions)
Biotinylated aβ42, by AnaSpec (1 mentions)
Plasmid maxi kit, by Qiagen (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Nis element, by Nikon (1 mentions)
4 protocols using anti aβ antibody 4g8
1
Quantifying Amyloid-beta and AICD in Gamma-Secretase Assays
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
DNA Sequencing and Aβ Peptide Analysis
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Reagents for DNA sequencing (Dye Terminator Cycle Sequencing FS Ready Reaction Kit) were purchased from Applied Biosystems (Foster City, CA). Synthesized primers were supplied by Hokkaido System Science (Sapporo, Japan). The plasmid purification kit (QIAGEN Plasmid Maxi Kit, QIAprep Spin Miniprep Kit) and the gel extraction kit (Wizard SV Gel and PCR Clean-Up System) were purchased from Qiagen and Promega (Tokyo, Japan), respectively. Aβ l-42 was supplied by the Peptide Institute (Osaka, Japan). DNA ligase (T4 DNA ligase), M13 KO7 helper phage, and anti-M13 KO7/HRP labeled antibody were purchased from GIBCO BRL (Carlsbad, CA). Anti-Aβ antibody (4G8) was purchased from COVANCE (CA).
4
Quantifying Amyloid-Beta Deposition in Mouse Brain
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Coronal slices of the right hemibrain were embedded in paraffin and cut (7μm); sections were de-waxed in xylene and hydrated through serial alcohols to water. After formic acid (80%) pre-treatment, sections were incubated overnight with anti-Aβ antibody (4G8, 1:4000; Covance). The primary antibody signal was detected with a biotinylated secondary antibody followed by horseradish streptavidin peroxidase and visualized with DAB. Amyloid deposition was quantified in mouse brain using Aβ immunostaining (4G8) and the staining intensity was evaluated semiquantitatively using a scale range from 0 to 5 by light microscopy. The assessment was conducted in two adjacent sections of the same brain area49 (link). A parallel quantification of the Aβ plaque load was performed using image analysis software (NIS-elements-Nikon)50 (link)51 (link)52 (link).
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