Homogenate samples of the left hippocampus and the cortex were prepared as described in a previous study [20 (link)]. The hippocampus or cortex was homogenized in tris-buffer solution (TBS) containing a protease inhibitor cocktail (BioVision, CA, USA) using a multi-beads shocker (Yasui Kikai, Osaka, Japan). The supernatant (first) was collected after centrifugation at 50,000×g for 20 min, and the pellet was homogenized again in TBS containing 1% Triton X-100 (Wako, Osaka Japan), and the supernatant (second) was collected after centrifugation at 50,000×g for 20 min. The total protein concentration of each supernatant was measured using the BCA Protein Assay Kit (Thermo-Scientific, Yokohama, Japan). The first supernatant was used to quantify soluble Aβ1-42 (Wako), phosphorylated tau (pS199, ThermoFisher Scientific, Yokohama, Japan), total tau (ThermoFisher Scientific), synaptophysin (LSBio, Seattle, WA, USA), BDNF (Promega, Madison, WI, USA), and IGF-1 (R&D Systems, Minneapolis, MN, USA) by ELISA and cytokines and chemokines by a Bio-Plex assay system (Bio-Rad, Hercules, CA, USA). The second supernatant was used to quantify insoluble Aβ1-42 (Wako) by ELISA.
Aβ1 42
Manufactured by Fujifilm
Sourced in Japan, United States
Aβ1–42 is a peptide fragment that is commonly used in research applications. It is a component of the amyloid-beta protein, which is associated with the pathogenesis of Alzheimer's disease. This product is intended for use in in vitro studies and laboratory experiments, and its core function is to serve as a research tool for investigating amyloid-beta biology and related disease mechanisms.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Multi beads shocker, by Yasui Kikai (3 mentions)
Aβ1 40, by Fujifilm (3 mentions)
Brain derived neurotrophic factor (bdnf), by Promega (2 mentions)
Bio plex assay system, by Bio-Rad (2 mentions)
Nerve growth factor (ngf), by Promega (1 mentions)
Mip 1α, by R&D Systems (1 mentions)
Anti aβ1 42, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Fujifilm (1 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Protease inhibitor cocktail, by Abcam (1 mentions)
Total tau, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Fujifilm (1 mentions)
Igf 1, by R&D Systems (1 mentions)
Spectramax plus 384, by Molecular Devices (1 mentions)
Tbs buffer, by Fujifilm (1 mentions)
Silica gel 60, by Kanto Chemical (1 mentions)
600 mhz nmr spectrometer, by Agilent Technologies (1 mentions)
8 protocols using aβ1 42
1
Hippocampus and Cortex Protein Quantification
2
Protein Preparation and Aggregation
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HEWL, h-Lz, and Aβ 1–42 were purchased from FUJIFILM Wako Pure Chemical Co. (Osaka, Japan). All other reagents were of biochemical grade.
3
Fermented Dairy Effects on Alzheimer's
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To evaluate the effects of fermented dairy products on Alzheimer’s disease, 3-month-old transgenic and wild-type female mice were fed a diet with or without fermented sample (2% w/w) for three months (n = 11 mice in each group). At the age of 6 months, the mice were euthanized and the brains were removed. The left hemisphere of the brain was homogenated in RIPA buffer (Wako) with a multi-beads shocker (Yasui Kikai, Osaka, Japan). After centrifugation at 500,000 × g for 20 min, the total protein concentration of the supernatant was measured with a BCA protein assay kit (ThermoScientific, Yokohama, Japan). To quantify Aβ1–42 (Wako), MIP-1α (R&D systems, MN, USA), TNF-α (eBiosciences, CA, USA), IL-1β (eBiosciences), synaptophysin (Life Science Inc., FL, USA), BDNF (Promega, WI, USA), GDNF (Promega), and NGF (Promega) in the hippocampus, an appropriate ELISA kit was used. The right hemispheres were fixed in 10% formalin (Wako) and analyzed immunohistochemically using the following specific antibodies: anti-Aβ1–42 (polyclonal, Invitrogen, CA, USA), anti-Iba-1 (polyclonal, Wako), and anti-MIP-α (polyclonal, R&D systems).
4
Plasma Aβ Quantification Protocol
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Plasma was collected at baseline, 4 weeks, and 8 weeks. Plasma samples from each time point were analyzed using enzyme-linked immunosorbent assays for Aβ1–40 and Aβ1–42 (Wako Pure Chemical Industries, Osaka, Japan) according to kit instructions. All samples were run in triplicate. The samples were read on a SpectraMax Plus 384 spectrophotometer and plate reader (Molecular Devices, Sunnyvale, CA, USA).
5
Quantification of Neuroinflammatory Markers
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To measure levels of cytokines, Aβ, and Tau, we homogenized the hippocampus of the left hemisphere in TBS buffer (Wako) with a multi-beads shocker (Yasui Kikai, Osaka, Japan). After centrifugation at 50,000× g for 20 min, we collected the supernatant. We measured the total protein concentration of each supernatant with a BCA protein assay kit (ThermoScientific, Yokohama, Japan). We assayed the supernatant to quantify soluble Aβ1–42 (Wako), Tau (ThermoScientific), and phosphorylated Tau (pTau, pS199, ThermoScientific) by ELISA and cytokines by a Bio-Plex assay system (Bio-Rad, Hercules, CA, USA).
6
Amyloid-beta Peptide Preparation Protocol
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Unless otherwise specified, all commercially
available compounds were used as provided without further purification.
Solvents for chromatography were of technical grade. Reactions were
monitored by thin-layer chromatography (TLC) carried out on silica
gel plates (TLC Silica Gel 60 F254, Merck). Column chromatography
was performed using Silica Gel 60 (spherical, 63–210 μm,
KANTO CHEMICAL). 1H and 13C nuclear magnetic
resonance (NMR) spectra were recorded with a JEOL JNMAL-400 (400 MHz)
NMR and a Varian 600 MHz NMR spectrometer, respectively, using acetone-d6 or dimethyl sulfoxide (DMSO)-d6 as the solvent and internal reference. Mass spectra
were measured with a JMS-700V (JEOL) mass spectrometer. Sib, the mixture
of silybin A and silybin B, was purchased from Sigma Aldrich, Inc.
Th-T and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were purchased from
Sigma-Aldrich, Inc. Aβ1–42 (hydrochloride
salt) was purchased from Wako Pure Chemical Industries, Ltd. Aβ1–42 (trifluoroacetic acid salt) was purchased from
Peptide Institute, Inc. Prior to experiments, Aβ1–42 was completely dissolved in HFIP using sonication. After dispensing
the solution into appropriate volumes, the solvent was evaporated
and the resultant Aβ1–42 was stored at −80
°C before use. Reagents for cell culture were used as provided
without further purification.
available compounds were used as provided without further purification.
Solvents for chromatography were of technical grade. Reactions were
monitored by thin-layer chromatography (TLC) carried out on silica
gel plates (TLC Silica Gel 60 F254, Merck). Column chromatography
was performed using Silica Gel 60 (spherical, 63–210 μm,
KANTO CHEMICAL). 1H and 13C nuclear magnetic
resonance (NMR) spectra were recorded with a JEOL JNMAL-400 (400 MHz)
NMR and a Varian 600 MHz NMR spectrometer, respectively, using acetone-d6 or dimethyl sulfoxide (DMSO)-d6 as the solvent and internal reference. Mass spectra
were measured with a JMS-700V (JEOL) mass spectrometer. Sib, the mixture
of silybin A and silybin B, was purchased from Sigma Aldrich, Inc.
Th-T and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) were purchased from
Sigma-Aldrich, Inc. Aβ1–42 (hydrochloride
salt) was purchased from Wako Pure Chemical Industries, Ltd. Aβ1–42 (trifluoroacetic acid salt) was purchased from
Peptide Institute, Inc. Prior to experiments, Aβ1–42 was completely dissolved in HFIP using sonication. After dispensing
the solution into appropriate volumes, the solvent was evaporated
and the resultant Aβ1–42 was stored at −80
°C before use. Reagents for cell culture were used as provided
without further purification.
7
Quantification of Amyloid-Beta Peptides
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The levels of Aβ1–40 and Aβ1–42 in both the soluble and insoluble fractions were determined using an ELISA kit (#294–64701, Aβ1–40 and #292–64501, Aβ1–42; Wako Pure Chemical Industries) in accordance with the manufacturer’s instructions. The snap-frozen brain were homogenized in ice-cold RIPA (radioimmunoprecipitation assay) buffer (Nacalai Tesque, Inc., Kyoto, Japan) containing 1% protease inhibitor cocktail and 1 × PhosSTOP (Roche Diagnostics GMbH, Basel, Switzerland), followed by centrifugation at 100,000 × g for 1 h at 4°C. The supernatant was collected for further analysis of soluble Aβ peptides. Then the pellet was extracted in ice-cold 70% (v/v) formic acid (Wako Pure Chemical Industries) followed by centrifugation at 100,000 × g for 1 h at 4°C. The supernatant was neutralized with a 20-fold dilution in 1M Tris buffer and analyzed for insoluble Aβ peptides. The protein concentration of individual fractions was measured by BCA assay (Thermo Fisher Scientific).
8
Quantification of Amyloid-Beta Proteins
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Aβ1-40, Aβ1-42, anti-Aβ ELISA kits, mouse anti-6xHis-tag antibody, anti-mouse HRP-conjugated antibody were purchased from Wako. Passive Lysis Buffer was ordered from Promega. Anti-6xHis-tag ELISA kit was from Genscript. pFUSE2ss-CLIg-hl2 plasmid was from Invivogen. Human Embryonic Kidney cells expressing the T7 polymerase (HEK 293-T7) were a kind gift from Pr. Huang and Pr Midoux.(26)
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