Total protein was extracted from LPS-stimulated cells treated with antibiotics by using 200 µL of radioimmunoprecipitation assay buffer (FUJIFILM Wako, Osaka, Japan) containing a protease inhibitor cocktail (Nakalai Tesque, Kyoto, Japan) and the lysates were clarified by centrifugation (15,000 rpm, 10 min, 4 °C). Protein concentration was determined using Pierce 660 nm Protein Assay Kit (Thermo Scientific, Rockford, USA). Samples containing 10 µg of protein were run on a 10% polyacrylamide gel and electrotransferred onto a membrane filter (Immobilon-P; Millipore, Bedford, MA, USA). The membrane was blocked Blocking One (Nacalai Tesque) for 30 min, followed by incubation at room temperature for 1 h with a rabbit polyclonal antibody (Cell Signaling, Danvers, MA, USA) phospho-NF-κB p65, NF-κB p65, phospho-ERK, ERK, phospho-p38, p38, TACE and phospho-TACE (Abcam, Cambridge, UK). The membrane was then incubated at room temperature for 30 min with horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G antibodies (GE Healthcare Bio-Science, Little Chalfont, England). Immunoreactive bands were visualized using enhanced chemiluminescence ImmunoStar LD (FUJIFILM Wako) and a FUSION-SOLO.7S.EDGE Chemilluminescence Imaging System (Vilber-Lourmat, 24 rue de Lamirault, 77090 Collégien, France). The data shown are representative of 3 independent experiments.
Pierce 660 nm protein assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Pierce 660 nm Protein Assay Kit is a colorimetric assay used for the quantitation of protein concentrations. It utilizes a dye-binding reagent that changes color in proportion to protein concentration, allowing for the measurement of total protein levels in a sample.
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Lab products found in correlation
Srebp2, by BD (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Procarta plex cell lysis buffer, by Thermo Fisher Scientific (3 mentions)
Odyssey classic imaging system, by LI COR (3 mentions)
Immunocap assay, by Thermo Fisher Scientific (3 mentions)
Z lle amc, by R&D Systems (2 mentions)
Suc llvy amc, by R&D Systems (2 mentions)
Spectramax m5, by Molecular Devices (2 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (2 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (2 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (2 mentions)
Chemidoc xr, by Bio-Rad (2 mentions)
Tissuelyser mechanical homogenizer, by Qiagen (2 mentions)
Safe lock tube, by Eppendorf (2 mentions)
P44 42 mapk erk1 2, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Hyblot cl autoradiography film, by Thomas Scientific (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Luminex xmap technology based magpix, by Thermo Fisher Scientific (2 mentions)
100 protocols using pierce 660 nm protein assay kit
1
Protein Extraction and Western Blot Analysis
2
Quantifying Proteasome Enzyme Activity
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To determine the magnitude of proteasome inhibition, we assayed for proteasome activity with blinding to treatment groups as previously described.22 Briefly, bilateral biceps muscles were homogenized in 20 mM of Tris‐HCl, pH 7.2, 0.1 mM of EDTA, 1 mM of 2‐mercaptoethanol, 5 mM of ATP, 20% of glycerol, and 0.04% of Nonidet P‐40.31 Following centrifugation, protein concentration was determined using the Pierce 660 nm protein assay kit (Thermo Scientific #22662). The caspase‐like activity of the 20S proteasome β‐1 catalytic subunit and chymotrypsin‐like activity of the β‐5 catalytic subunit were assayed with 25 μg total protein per muscle, through detection of the 7‐amino‐4‐methylcoumarin (AMC) labeled fluorogenic peptide substrates Z‐LLE‐AMC (S‐230, Boston Biochem) and Suc‐LLVY‐AMC (S‐280, Boston Biochem), respectively. Endpoint fluorescence was measured at 380 nm/460 nm on a SpectraMax M5 microplate reader (Molecular Devices), and relative fluorescence units were then calculated per μg protein.
3
Protein Extraction from Frozen Cells
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Frozen cells were suspended in M-PER mammalian protein extraction reagent (Thermo Fisher Scientific) supplemented with Protease Inhibitor Cocktail for Use with Mammalian Cell and Tissue Extracts (Nacalai Tesque), and lysed by incubation for 30 min on ice. Cell lysates were centrifuged at 14,000 × g for 15 min at 4 °C, and the supernatant was collected as protein extract. The protein concentration was determined by using a Pierce 660 nm Protein Assay kit (Thermo Fisher Scientific) and bovine serum albumin as a protein standard according to the manufacturer’s instructions.
4
Quantitative Analysis of Tissue Biomarkers
5
Western Blot Protein Extraction and Analysis
6
Protein Expression Analysis of BMSCs
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After various treatments, the BMSCs were collected in PBS with 1% Halt™ Protease and Phosphatase Inhibitor (Thermo, IL) and stored at -80°C until use. Total protein concentrations in the lysates were determined using the Pierce™ 660nm protein assay kit (Thermo, IL). Then, 10-μg/lane proteins were loaded on SDS-PAGE gels. After blocking, the PVDF membrane (Bio-Rad) was incubated overnight at 4°C with one of the following antibodies: rabbit anti-superoxide dismutase (SOD) 1 antibody (1:2000; LifeSpan, WA), goat anti-malondialdehyde (MDA) antibody (1:2500; LifeSpan), rabbit anti-NF-κB p65, and anti-phospho-NF-κB p65 (Ser536) antibodies (1:1000; Cell Signaling, MA). The HRP-conjugated secondary antibodies were from Jackson ImmunoResearch (PA). Signals were detected using ECL Prime Western Blotting Detection Reagent (GE Healthcare, Buckinghamshire, UK) and ChemiDocXRS+ (Bio-Rad), then quantified densitometrically by the software Image Lab 4.1 (Bio-Rad).
7
Western Blot Analysis of Sult1a1 in Mouse Cerebrum
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Mice cerebrums (n = 6) from 2 litters (3 females per litter) with different parents were homogenized in a 9-fold volume of ice-cold brain lysis buffer (50 mM Tris-HCl, pH 7.4, 2.0 mM EDTA, 8.5% sucrose and 10 mM β-mercaptoethanol) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentration of the samples was measured by a Pierce™ 660 nm protein assay kit (Thermo Fisher Scientific). The same levels of protein in each sample were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein was then transferred onto polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skim milk in Tris-buffered saline (TBS, 150 mM NaCl, 50 mM Tris-HCl, pH 7.4) for 30 min and incubated with rabbit anti-Sult1a1 antibody (1:500, bs-6283R, Bioss Antibodies, Beijing, China) overnight at room temperature. Then the membrane was rinsed three times with TBST (TBS containing 0.05% Tween-20) and incubated with peroxidase affinipure goat anti-rabbit IgG (111-035-144) (Jackson ImmunoResearch, West Grove, PA, USA) for 2 hr at room temperature, and then developed with an Enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific) and exposed to X-ray film (Carestream Health, White City, OR, Canada). The intensity of bands was quantified using Multi Gauge software (Fuji Film, Tokyo, Japan).
8
Multiplex Cytokine Quantification in Tissue
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The protein concentrations for tissue extracts were determined using the Pierce 660nm Protein Assay Kit (Thermo Scientific Inc., NY, USA). Samples were thawed at room temperature and vortexed to ensure well-mixed sample. Multiple cytokine analysis kits (IL-5, IL-17A, IL-23, IFN-γ, CXCL-8, and CCL-11) were obtained from R&D systems (Cat. No. LMSAHM) and data were collected using Luminex 100 (Luminex, Austin, TX, USA). Data analysis was performed using the MasterPlex QT version 2.0 (MiraiBio, Alameda, CA). All assays were run in duplicate according to the manufacturers’ protocol. Sensitivity of each cytokine is as follows: IL-5 (0.5 pg/mL), IL-17A (1.8 pg/mL), IL-23 (11.4 pg/mL), IFN-γ (0.4 pg/mL), CXCL-8 (1.8 pg/mL), and CCL-11 (14.6 pg/mL), All the protein levels in tissue homogenate were normalized to the concentration of total protein (mg/mL) [17 (link)].
9
Isolation and Characterization of ARPE-19 Cell Mitochondria
10
Liver Protein Extraction Protocol
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For each group, six liver samples (two from three tanks) were subjected to protein extraction according to Ghisaura et al. (2019) (link) with some modifications. Briefly, a small portion of each tissue (200 mg) was placed in a 2 ml Eppendorf safe-lock tube (Eppendorf, Hamburg, Germany) and immersed in lysis buffer (0.065 M Tris-HCl pH 6.8, 1% dithiothreitol, 2% sodium dodecyl sulfate) in 1:4 (w/v) ratio, plus protease inhibitor cocktail (protease inhibitor cocktail for General Use, Sigma-Aldrich, Saint Louis, MO, United States) as indicated in the manufacturer instructions. Samples were then subjected to three cycles of bead beating for 5 min at 30 oscillations/s in a TissueLyser mechanical homogenizer (Qiagen, Hilden, Germany), and frozen between homogenization cycles to facilitate tissue disruption and prevent excessive sample heating. Samples were then centrifuged for 15 min at 14,000 × g at 4°C. The final supernatants were collected as the liver protein extracts, diluted 1:50 in lysis buffer and quantified with the Pierce 660 nm Protein Assay Kit (Thermo Scientific - Rockford, IL, United States). Protein extracts were stored at −80°C until use.
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