Post-mortem brain samples from the frontal cortex from FAD APPSwe (n = 4, ages between 56 and 66, post-mortem time between 24 and 40 h) and non-demented controls (n = 4, ages between 67 and 82, post-mortem time between 9 and 27 h) were used to assess MERCS-related proteins (further description of patients in Supplementary Table S1 ). Frozen tissue was cut in pieces while kept cold on ice and homogenized with 5 cm3 glass-Teflon homogenizer (1200 rpm) in digestion solution (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate (DOC), 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris pH = 7.5 (all chemicals from Sigma-Aldrich), 1x mammalian ProteaseArrest (G-Biosciences, St. Louis, MO, USA #786-433), Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich, #P0044) and 1x Benzonase (Merck, Darmstadt, Germany, #70664)). After 10 min of incubation, a quick homogenization was performed with the same Teflon homogenizer (1200 rpm) and the homogenate centrifuged at 7000× g, for 10 min at 4 °C to remove debris. Supernatant was collected and protein concentration determined by the Pierce™ bicinchoninic acid assay BCA Protein Assay Kit (ThermoFisher, #23225). All the steps were performed on ice.
Pierce bicinchoninic acid assay bca protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce bicinchoninic acid (BCA) assay kit is a colorimetric detection method for quantifying total protein concentration in a solution. The kit uses a combination of the biuret reaction and the reduction of copper ions to provide a sensitive and stable color change that can be measured spectrophotometrically.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Probe sonicator, by Emerson (1 mentions)
6 well plate, by Sarstedt (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Ldl vldl purification kit, by Cell Biolabs (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
Pierce albumin depletion kit, by Thermo Fisher Scientific (1 mentions)
Amplex red acetylcholine acetylcholinesterase assay kit, by Thermo Fisher Scientific (1 mentions)
M per buffer, by Thermo Fisher Scientific (1 mentions)
Serum replacement solution, by Thermo Fisher Scientific (1 mentions)
0.22 μm filter, by Merck Group (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Nanosight system, by Malvern Panalytical (1 mentions)
Actin, by Cell Signaling Technology (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
0.2 μm nitrocellulose membrane, by Bio-Rad (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Cell Signaling Technology (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
14 protocols using pierce bicinchoninic acid assay bca protein assay kit
1
Quantification of MERCS Proteins in FAD
2
Cell Viability Assay Protocol
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B16–F10 and LLC2 were seeded into 6-well plates (Sarstedt) at a density of 100 000 per well and grown for 24 h. Then cells were treated with IC50 concentration of each drug and corresponding concentration of DMSO (Fig. 1 ). Treatments were conducted in triplicates for each drug and cell lines. After 48 h of treatment, cells were washed two times with PBS, whereupon lysis buffer (1% SDS, 8 M urea, 50 mM Tris pH 8.5) was added on top of the cells. Cells were scraped and collected into tubes and then sonicated using a probe sonicator (Branson) for 45s (3s/3s pulse, 30% amplitude). Protein concentration was measured in each sample using Pierce bicinchoninic acid assay (BCA) protein assay kit (Thermo Fischer Scientific) according to the manufacturer's protocol.
3
Amyloid-β Detection and Quantification
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Transfection reagent Lipofectamine 3000 (L3000008) was purchased from Thermo Fisher Scientific. Dulbecco’s Modified Eagle’s Medium (41965039, high glucose) was purchased from Thermo Fisher Scientific. Fetal bovine serum (10270-106) was purchased from Thermo Fisher Scientific. Poly-d -lysine precoated 8-well plates (354632) were purchased from Corning. Duolink In Situ Mounting Medium with DAPI (DUO82040) was purchased from Sigma-Aldrich; 10× RIPA buffer (20–188) was purchased from Millipore. γ-Secretase inhibitor L-685,485 (2627) was purchased from Tocris. NuPage 4–12% polyacrylamide Bis-Tris gel (NP0321BOX) and 10× Tris buffered saline (J60764, pH 7.4) were purchased from Thermo Fisher Scientific. Nitrocellulose blotting membranes (10600004) were purchased from GE Healthcare Life Science. Duo prestained protein ladder (928-60000) was purchased from LI-COR Biosciences. Pierce bicinchoninic acid assay (BCA) protein assay kit (23225) was purchased from Thermo Fisher Scientific. Human Amyloid-β 1–42 ELISA kit (27719) was purchased from IBL.
4
Isolation and Characterization of VLDL
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VLDL was isolated from pooled serum from patients and HCs using the using LDL/VLDL Purification Kit (Ultracentrifugation Free, CELL BIOLABS, San Diego, California, USA) according to manufacturer’s instructions. Purified LDL/VLDL was dialysed in PBS in Amicon Ultra centrifugal filter units (MILLIPORE, Hertfordshire, UK). A Pierce™ Bicinchoninic Acid assay (BCA) Protein Assay Kit (ThermoFisher) was used to quantify the isolated LDL/VLDL (Apolipoprotein B protein expression). Isolated lipoproteins were filtered through a 0.22-μm filter and stored at 4 °C for a maximum of 6 weeks. 1 × 106 PBMCs were cultured with 10% isolated VLDL (concentration proportionate to physiological levels of the original pooled serum groups) in RPMI-1640 medium 48 h followed by lipid analysis by flow cytometry.
5
Albumin Depletion of Serum Samples
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Before serum albumin depletion, protein concentration was carefully assessed in all samples by a Pierce bicinchoninic acid assay (BCA) Protein Assay Kit (ThermoFisher Scientific, Rodano, Italy). Albumin depletion was performed according to the manufacturer’s instructions using the Pierce Albumin Depletion Kit (ThermoFisher Scientific). Briefly, 400 µL of Pierce depletion resin (corresponding to 200 µL of settled resin) was transferred to a spin column, and the column was centrifuged and washed with 200 µL of binding/wash solution. After centrifuging at 12,000× g for 1 min, 50 µL of serum sample was added to the tube. The tube was incubated for 2 min at room temperature and centrifuged at 12,000× g for 1 min. To release unbound proteins, we washed spin tubes 3 times with 50 µL of binding/wash solution and then centrifuged them at 12,000× g for 1 min.
6
Ileal Mucosal Protein Extraction and AchE Activity
7
Isolation and Characterization of ADSC-derived Exosomes
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We isolated ADSC-derived Exos when cells reached 80%-90% confluence. Our team rinsed ADSCs from various groups with phosphate-buffered saline (PBS), and then cultured them in FBS-free endothelial cell growth medium (EGM)-2MV, which was supplemented with 1 × serum replacement solution (PeproTech, Rocky Hill, NJ, United States) for another 2 d. Then, we collected conditioned culture medium and centrifuged it at 300 × g for 10 min to remove cells and at 2000 × g for another 10 min to remove apoptotic cells and cellular debris. Following centrifugation at 10000 × g for 30 min, we filtered the supernatant through a 0.22 μm filter (Millipore, Billerica, MA, United States), then transferred 15 mL supernatant to the Amicon Ultra-15 Centrifugal Filter Unit (100 kDa) and centrifuged it at 4000 × g to concentrate to approximately 1 mL. The ultrafiltration unit was washed twice with PBS centrifuged it again at 100000 × g, and the supernatant was aspirated. All processes were conducted at 4 °C. We resuspended the Exo pellets obtained in 500 μL PBS. Finally, the Exo protein content was evaluated using the Pierce bicinchoninic acid assay (BCA) Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, United States). We stored Exos at -80 °C until subsequent use for experiments and identified Exos by western blotting and transmission electron microscopy.
8
Quantifying Epigenetic Repression Markers
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Protein lysates were prepared using radioimmunoprecipitation assay (RIPA) lysis buffer (0.1% sodium dodecyl sulfate, 1% NP‐40, 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris PH 7.5, 150 mM NaCl, 0.25% deoxycholate) with protease and phosphatase inhibitors (Roche, Welwyn Garden City, UK). Protein concentration was determined using the Pierce bicinchoninic acid assay (BCA) Protein Assay Kit (ThermoFisher Scientific, Hemel Hempstead, UK). Following sodium dodecyl sulfate polyacrylamide gel electrophoresis electrophoresis and subsequent immunoblotting, bound anti‐EZH2 antibody (1:1000), anti‐histone H3 tri‐methyl K27 (1:1000), anti‐β‐tubulin (1:1000), was detected by developing film from Western blot analysis substrate (Promega, Southampton, UK).
9
Quantification of Extracellular Vesicles
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Quantification of extracellular vesicles based on proteins was performed in a 96-well plate using the Pierce Bicinchoninic acid assay (BCA) protein assay kit (Thermo Fisher, Waltham, Massachusetts) according to the manufacturer’s protocol. A Spark Tecan Reader (Tecan Trading AG, Maennedorf, Switzerland) was used for the read out of the plate. The results were used to determine the amount of extracellular vesicles needed for injection (100 μg). Quantification of extracellular vesicles based on the number of particles, as well as size determination, was achieved using a NanoSight system (Malvern Instruments, Malvern, UK) with a green 532-nm laser and an Andor CCD camera.
10
Protein Extraction and Western Blot Analysis
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Cell samples were washed twice with ice-cold phosphate-buffered saline (PBS) prior to harvesting in radioimmunoprecipitation assay (RIPA) buffer (Cell Signaling, Danvers, MA) with 1% phenylmethanesulfonyl fluoride protease inhibitor (PMSF, Cell Signaling) and phosphatase inhibitor cocktail 2 (P5726, Sigma). Cells were lysed with sonication and vortexing, and cell debris was pelleted with centrifugation at 14,000 RPM for 15 minutes. Protein concentration of the supernatant was determined with Pierce bicinchoninic acid assay (BCA) protein assay kit (ThermoFisher, Waltham, MA). Equal amounts of protein (25 μg) were separated on polyacrylamide gels and transferred to 0.2 μm nitrocellulose membranes (Bio-Rad, Hercules CA). Membranes were probed with antibodies for Actin, ATF4, and GPT2 (Actin Cell Signaling #4970, 1:4000; ATF4 Cell Signaling #11815, 1:1000; GPT2 ThermoFisher #16757-1-AP, 1:1000) overnight in 5% nonfat dry milk. Membranes were incubated with secondary antibodies (LiCor # 926-68073, 1:10,000) for one hour at room temperature. Protein was detected using an Odyssey CLx imaging system (Li-Cor, Lincoln, NE).
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