The Aβ1-40 peptide was obtained from Sigma-Aldrich (St. Louis, MO, USA). STF-083010, a specific IRE1α I endonuclease inhibitor, was bought from Sigma-Aldrich and was prepared fresh in a dark room with DMSO for a 25 mM stock solution. The RNA Fast Isolation kit and M-MLV Reverse Transcriptase were obtained from BioTeke Corporation (Beijing, China). The SYBR Green PCR Kit was purchased from Solarbio Science & Technology (Beijing, China), and the Caspase-2 cellular activity assay kit was purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The LDH Activity Assay Kit was purchased from Abcam. The miR-34a mimic oligonucleotide and control RNA were purchased from GenePharma (Shanghai, China). The Lipofectamine™ 2000 reagent was obtained from Invitrogen Life Technologies (Carlsbad, CA, USA). Antibodies for the Western blot assay were purchased as follows: XBP1, IRE1α, and p-IRE1α antibodies (Abcam, Cambridge, UK) and Caspase-2 and β-actin antibodies (Sigma-Aldrich, St. Louis, MO, USA). All culture media and fetal bovine serum (FBS) were obtained from Gibco (Carlsbad, CA, USA). Unless specifically mentioned, all other reagents were purchased from Sigma-Aldrich.
Aβ1 40 peptide
Manufactured by Merck Group
Sourced in United States
Aβ1-40 peptide is a synthetic peptide that corresponds to the first 40 amino acids of the amyloid-beta protein. It is commonly used in research applications to study the structure, aggregation, and biological effects of amyloid-beta, which is associated with Alzheimer's disease.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by BioTeke (1 mentions)
Stf 083010, by Merck Group (1 mentions)
Sybr green pcr kit, by Solarbio (1 mentions)
Control rna, by GenePharma (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Acetylthiocholine iodide, by Merck Group (1 mentions)
5 5 dithiobis 2 nitrobenzoic acid dtnb, by Merck Group (1 mentions)
Tlc plate, by Merck Group (1 mentions)
Sh sy5y cell, by American Type Culture Collection (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Human insulin, by Merck Group (1 mentions)
Aβ1 40, by Merck Group (1 mentions)
Thioflavin t, by Merck Group (1 mentions)
6 protocols using aβ1 40 peptide
1
Investigating Amyloid-Beta Induced Apoptosis
2
Preparation of Amyloid-beta Oligomers
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Scrambled Aβ, oligomeric Aβ1-42, or Aβ1-40 was prepared as previously described (Du et al., 2010 (link); Rui et al., 2006 (link); Vossel et al., 2010 (link)). Aβ1-42 or Aβ1-40 peptide (Sigma) was diluted in 1,1,1,3,3,3-hexafluoro-2-propanol to 1 mM using a glass gas-tight Hamilton syringe with a Teflon plunger. The clear solution was then aliquoted in microcentrifuge tubes, followed by evaporation in the fume hood over night at RT, and it was then dried under vacuum for 1 hr in a speedVac (DNA Vap, Labnet, Edison, NJ). Peptide film was diluted in DMSO to 5 mM and sonicated for 10 min in bath sonicator. The peptide solution was resuspended in cold TBS buffer to 100 μM and immediately vortexed for 30 s; the solution containing monomeric Aβ was then incubated at 4°C for 24 hr to form oligomeric Aβ before applying to in vitro binding assays.
3
Amyloid-beta Peptide Activity Assay
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Aβ (1–40) peptide, acetylthiocholine iodide (AChI), and 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich USA. All other chemicals used in the study were of analytical grade. Solutions of the drug and chemicals were freshly prepared before use.
4
Neuronal Cell Culture and Assays
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Dulbecco’s modified eagle’s medium and FBS were purchased from Gibco (United States), and SHSY5Y cells were from ATCC. 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), 2′,7 di-chloro fluorescein di-acetate (DCFD), SDS, TBA, TLC plates, Aβ1-40 peptide, acetylcholine iodide (ACTI) and MTT were from (Merck-Sigma Aldrich) and other chemicals were purchased from Hi-media.
5
Preparation of Amyloid-β Oligomers
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The oligomer Aβ1-40 peptide (Sigma, St. Louis, USA) was prepared according to the manufacturer's instructions. In short, the freeze-dried Aβ1-40 peptide was dissolved in deionized distilled water (6 μg/μl). Before incubating at 37°C for 4 days, the Aβ1-40 oligomer was diluted with phosphate-buffered saline to a concentration of 1.5 μg/μl and stored at -20°C until use. Aβ40-1 peptide (Sigma, St. Louis, USA) was used as a negative control for Aβ1-40, and Aβ40-1 was handled in the same way as Aβ1-40.
6
Amyloid Aggregation Assay Protocols
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Ubiquitin from bovine erythrocytes, recombinant Human Insulin and Thioflavin T were purchased from Sigma Aldrich and used without further purification. Aβ(1-40) was purchased from Biopeptide and pre-treated as previously reported 64 .
All experiments on UBQ samples were performed in 20 mM sodium phosphate buffer, pH 6.5. The sample was freshly prepared and filtered through 0.20 μm filters (17761, Sartorious), just before the measurements. Different protein concentrations had been used: 0.5 mg/ml, 0.7 mg/ml and 1.8 mg/ml. Protein concentration was spectrophotometrically determined using a molar extinction coefficient of 1254 dm 3 mol -1 cm -1 at 280 nm 31 . Human Insulin powder (Sigma-Aldrich) and Aβ (1-40) peptide (prepared as described in 65 ), were dissolved in 20mM sodium phosphate buffer, pH 6.5.
Protein concentration of Human Insulin and Aβ(1-40) peptide were estimated by means of absorbance measurements using a molar extinction of 1.067 cm -1 (mg/ml) -1 at 276 nm 66 , and of 1390 M -1 cm -1 at 276nm, respectively 67 .
In order to obtain aggregates, 0.2 mg/ml Human Insulin in 20 mM sodium phosphate buffer, pH 6.5, was incubated at 60°C stirring at500 rpm for 24 hours, and 0.2 µM Aβ(1-40) in 20 mM sodium phosphate buffer, pH 6.5, was incubated at 37 degrees with stirring 500 rpm for 24 hours.
Resulting aggregates were found to be positive to Thioflavin T test.
All experiments on UBQ samples were performed in 20 mM sodium phosphate buffer, pH 6.5. The sample was freshly prepared and filtered through 0.20 μm filters (17761, Sartorious), just before the measurements. Different protein concentrations had been used: 0.5 mg/ml, 0.7 mg/ml and 1.8 mg/ml. Protein concentration was spectrophotometrically determined using a molar extinction coefficient of 1254 dm 3 mol -1 cm -1 at 280 nm 31 . Human Insulin powder (Sigma-Aldrich) and Aβ (1-40) peptide (prepared as described in 65 ), were dissolved in 20mM sodium phosphate buffer, pH 6.5.
Protein concentration of Human Insulin and Aβ(1-40) peptide were estimated by means of absorbance measurements using a molar extinction of 1.067 cm -1 (mg/ml) -1 at 276 nm 66 , and of 1390 M -1 cm -1 at 276nm, respectively 67 .
In order to obtain aggregates, 0.2 mg/ml Human Insulin in 20 mM sodium phosphate buffer, pH 6.5, was incubated at 60°C stirring at500 rpm for 24 hours, and 0.2 µM Aβ(1-40) in 20 mM sodium phosphate buffer, pH 6.5, was incubated at 37 degrees with stirring 500 rpm for 24 hours.
Resulting aggregates were found to be positive to Thioflavin T test.
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