Aβ peptides (AnaSpec, Fremont, CA, USA) were dissolved at a concentration of 1 mg/mL in hexafluoroisopropanol (HFIP). Peptides were then mixed at different molar ratios. HFIP was evaporated in a SpeedVac without heating for 15 min. Peptides and mixtures were kept on ice and reconstituted in 50 mM Tris–HCl, 1 mM EDTA. Peptides (final concentration: 10 μM) were added to thioflavin T (final concentration: 2.5 μM; AnaSpec) in a black 96-well plate. Aggregation was monitored on a SpectraMax M5 plate reader (Molecular Devices, Inc.) at a constant temperature of 25 °C using excitation/emission of 440 nm/480 nm. Readings were recorded in triplicate every 10 min for a period of 18 h.
Aβ peptide
Manufactured by AnaSpec
Sourced in United States
Aβ peptides are synthetic peptides derived from the amyloid-beta protein. They are commonly used in research applications to study the pathology of Alzheimer's disease and other neurodegenerative disorders.
Automatically generated - may contain errors
Lab products found in correlation
Thioflavin t, by AnaSpec (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Hilyte555 aβ42, by AnaSpec (1 mentions)
Acetylthiocholine iodide atc, by Merck Group (1 mentions)
Infinite m200 pro multimode reader, by Tecan (1 mentions)
Equine buche, by Merck Group (1 mentions)
Hydrochloric acid (hcl), by Merck Group (1 mentions)
Cell culture slides, by MatTek (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Abcam (1 mentions)
3 aminopropyltriethoxysilane, by Merck Group (1 mentions)
Thioflavin t tht, by Thermo Fisher Scientific (1 mentions)
Cell fractionation kit, by Cell Signaling Technology (1 mentions)
Tween 20, by Merck Group (1 mentions)
1 2 dioleoyl sn glycero 3 phosphoethanolamine, by Avanti Polar Lipids (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
6 protocols using aβ peptide
1
Amyloid-beta Peptide Aggregation Kinetics
2
Preparation of Amyloid-Beta Peptides
3
Aβ Fibrillization and Cell Treatment
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Aβ peptides in powder form (Anaspec) were initially resuspended in 1% NH4OH for 15min to dissolve any pre-formed aggregates per manufacturer instructions, and then diluted to a final concentration of 0.05% NH4OH in water prior to fibrillization. Aβ peptides were converted to the fibrillar form of Aβ by incubating monomeric human Aβ(1-42) (cat. no: AS-20276) or human Hilyte Fluor 555 labeled Aβ(1-42) (cat. no: AS-60480-01) at 220μM in water at 37°C for 3days prior to use as previously described (34 (link)). Hilyte Fluor is more photostable than the classic fluorescent dyes. They are also highly fluorescent over a broad pH range with little pH sensitivity (Anaspec). Treatment with 1µM Aβ was completed in serum free media for 1h before washing away excess Aβ and adding full media.
4
Amyloid-beta Solubilization and Neuronal Exposure
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Amyloid-beta (Aβ) peptides were purchased from AnaSpec and solubilized in a manner analogous to previous protocols (Stine et al., 2003) . Briefly, lyophilized peptides were dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to generate a 1 mM solution. The Aβ-HFIP solution was aliquoted into PCR tubes and evaporated to dryness by placing the open PCR tubes in a chemical fume hood overnight followed by further concentration on a vacuum concentrator. The dried peptide aliquots were then stored over desiccant in glass jars at -20 °C.
Before addition to the cultures, the dried peptides were re-dissolved to 1 mM in DMSO, vortexed, and sonicated for 10 minutes. Further dilutions were made with DMSO to 50-500 μM. At 8 DIV, the DMSO stocks were diluted 1:500 in fresh Neurobasal media containing 2% B-27 supplement and 1% GlutaMax (NB++). The Aβ-NB++ solutions were then added to an equal amount of conditioned media on the coverslips to give a total dilution of 1:1000 for Aβ:Media. The cultures were grown in the Aβ-NB++ solution for 7 days and loaded with dye and imaged at 15 DIV. For vehicle controls, an equal amount of DMSO containing no Aβ was added to the NB++.
Before addition to the cultures, the dried peptides were re-dissolved to 1 mM in DMSO, vortexed, and sonicated for 10 minutes. Further dilutions were made with DMSO to 50-500 μM. At 8 DIV, the DMSO stocks were diluted 1:500 in fresh Neurobasal media containing 2% B-27 supplement and 1% GlutaMax (NB++). The Aβ-NB++ solutions were then added to an equal amount of conditioned media on the coverslips to give a total dilution of 1:1000 for Aβ:Media. The cultures were grown in the Aβ-NB++ solution for 7 days and loaded with dye and imaged at 15 DIV. For vehicle controls, an equal amount of DMSO containing no Aβ was added to the NB++.
5
Amyloid Inhibition Assay with AChE
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Recombinant human AChE (2419 U mg−1), expressed in human embryonic kidney (HEK) 293 cells, lyophilised powder (E.C. No. 3.1.1.7), equine BuChE (≥500 U mg−1), acetylthiocholine (ATC) iodide, butyrylthiocholine (BTC) iodide, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), donepezil, tacrine hydrochloride (A79922), propidium iodide (P4170), dimethyl sulfoxide (DMSO), methanol (MeOH) and ethanol (EtOH), di-sodium hydrogen orthophosphate dodecahydrate (Na2HPO4·12H2O), sodium dihydrogen orthophosphate (NaH2PO4·2H2O), sodium hydroxide (NaOH), thioflavin T, glycine, and hydrochloric acid (HCl) 37% were purchased from Sigma-Aldrich (St. Louis, MO, USA). β-Amyloid (Aβ) peptide (1–40) and Aβ peptide (1–42), lyophilised from HFIP solution were purchased from Anaspec (Fremont, CA, USA). The chemicals were of analytical grade. Instruments used included a UV/fluorescence spectrophotometer and multiplate reader, Infinite® M200 PRO multimode reader (Tecan Group Ltd., Männedorf, Switzerland), workstation and molecular modelling software.
6
Fluorescent Amyloid-Beta Nanoparticle Synthesis
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Tetraethyl orthosilicate, poly(N-isopropylacrylamide), ammonium hydroxide, dimethyl formamide (DMF), fluorescein isothiocyanate (FITC), Tween 20, 3-aminopropyl triethoxy silane (Sigma-Aldrich, St. Louis, MO), Calcein AM (CAM) (MilliporeSigma, Burlington, MA), polystyrene (PS) spheres (PolySciences, Warrington, PA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), rhodamine-labeled 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (Rh-PE) (Avanti Polar Lipids, Alabaster, AL), Opti-MEM (OM) reduced serum media, phosphate saline buffer (PBS), Thioflavin T (ThT) (Thermo Fisher Scientific, Waltham, MA), cell culture slides (MatTek, Ashland, MA), Aβ peptide (AnaSpec, Fremont, CA), biotinylated Aβ antibody (Mabtech, Stockholm, Sweden), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Abcam, Cambridge, UK), and Cell Fractionation Kit by Cell Signaling Technology (Danvers, MA) were used.
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