Curcumin was purchased from Plamed Green Science Limited (China). Endoxifen was purchased from Tocris Bioscience (USA). Beta estradiol, glutaraldehyde, poly-L-lysine were purchased from Sigma-Aldrich Ltd (Singapore). Dimethylsulfoxide (DMSO) was purchased Vivantis (Malaysia), DMEM-high glucose, FBS heat-inactivated, Penicillin/Streptomycin, Fungizone, were purchased from Biowest (USA). MTS assay kit was purchased from Promega (USA). High Pure RNA isolation kit, Transcriptor First Strand cDNA Synthesis kit, FastStart DNA master SYBR Green I were purchased from Roche (USA). Primers for E-cadherin, TGF-β1, vimentin and β-actin were purchased from First-base (Singapore), Human TGF-β1 ELISA kit and Coomassie Plus (Bradford) Assay Kit were purchased from invitrogen (USA).
Coomassie plus bradford assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Coomassie Plus (Bradford) Assay Kit is a colorimetric assay used to quantify total protein concentration in a sample. The kit utilizes Coomassie Brilliant Blue G-250 dye, which binds to proteins and results in a color change that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (3 mentions)
Jy92 iin sonicator, by Ningbo Scientz Biotechnology (3 mentions)
Heat inactivated fbs, by Biowest (2 mentions)
High pure rna isolation kit, by Roche (2 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (2 mentions)
Penicillin streptomycin, by Biowest (2 mentions)
Dmem high glucose, by Biowest (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Endoxifen, by Bio-Techne (1 mentions)
β estradiol, by Merck Group (1 mentions)
Faststart dna master sybr green 1, by Roche (1 mentions)
Tgf β1 human elisa kit, by Thermo Fisher Scientific (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Mts assay kit, by Promega (1 mentions)
Acetaldehyde, by Merck Group (1 mentions)
Sorafenib, by Santa Cruz Biotechnology (1 mentions)
32 protocols using coomassie plus bradford assay kit
1
Curcumin and Endoxifen Cytotoxicity Evaluation
2
Investigating Hepatic Stellate Cell Modulation
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Human hepatic stellate cells, LX-2, were purchased from Millipore (USA, Cat No. SCC064). Sorafenib and alfa mangostin were obtained from Santa Cruz Biotechnology (USA). Acetaldehyde, dimethylsulfoxide (DMSO), and TGF-β1 ELISA kit were from Sigma Aldrich (Singapore). DMEM-high glucose, FBS-heat inactivated, Penicillin/Streptomycin, and Fungizone were from Biowest (USA). High pure RNA isolation kit and Transcriptor First Strand cDNA synthesis kit were purchased from Roche (USA). Thunderbird SYBR qPCR Mix was purchased from Toyobo (Japan). Cell lysis buffer and Coomassie Plus (Bradford) assay kit were obtained from Invitrogen (USA). Primers were purchased from First Base (Singapore). Alpha smooth muscle actin antibody (PA5-19465) was purchased from Thermo Fisher (USA), GAPDH antibody was purchased from Santa Cruz Biotechnology (USA), and anti-rabbit IgG, HRP-linked antibody, ERK1/2, phospho-ERK1/2 rabbit mAb were obtained from Cell Signaling Technology (USA).
3
Isolation of Extracellular Vesicles from Reproductive Fluids
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EVs were isolated from oviductal and uterine flushings according to the protocol of serial ultracentrifugation described by Théry et al. (2006) (link) and Almiñana et al. (2017) (link) with some modifications. Briefly, each experimental group’s flushings from the oviducts and uteri were centrifuged at 300 × g for 15 min at 4°C to remove epithelial cells. The supernatant was collected and transferred to a new tube, then centrifuged at 2,000 × g for 10 min at 4°C to remove cellular debris. The supernatant was then ultracentrifuged at 100,000 × g for 70 min at 4°C (Beckman Coulter Optima L-100 XP ultracentrifuge with 70ti rotor) to pellet the EVs. The supernatant was discarded, and the pellet was resuspended in 3.5 mL PBS and ultracentrifuged again under the same conditions. The pellet was resuspended in a final volume of 300 µL PBS and aliquoted to 100 µL. One aliquot of EVs suspensions was analyzed fresh for protein concentration using the Coomassie Plus Bradford assay kit (23238, Fisher Scientific™, Waltham, MA, United States of America) according to the manufacturer’s protocol. The same amount of protein was processed and analyzed in each sample within the same type of reproductive fluid, OF or UF, corresponding to the sample with the lowest total amount of protein. The rest of the aliquots were stored at −80°C until further analysis.
4
Measurement of Cellular Signaling Factors
5
Evaluating Subfraction 5 Cytotoxicity on HepG2 Cells
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HepG2 cells were seeded into a 102 cm dish at a density of 1 × 106 cells/dish and incubated with various concentrations of subfraction 5 (0, 50, 100, 200, and 400 μg/mL) and 5-FU (25 μg/mL) for 24 h. The cells were harvested by cell scraping, rinsed with ice-cold PBS twice, and centrifuged at 3,000 rpm for 5 min. Pellet cells were lysed in a cold modified RIPA buffer with a protease inhibitor cocktail and then vortexed for 20 min at 4 °C. After centrifugation at 12,000 rpm for 20 min at 4 °C, the supernatant was collected to measure the total protein concentration using the Coomassie Plus (Bradford) Assay Kit (Thermo Science, Rockford, IL, USA). The protein samples were isolated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes by electroblotting. Next, they were blocked with 5% bovine serum protein (BSA) in TRIS-buffered saline with Tween 20 (TBST) for 1 h and soaked in primary antibodies (1 : 1000 dilutions) overnight in a 4 °C microplate shaker. The membranes were washed with TBST three times subsequently dipped in secondary antibodies for 1 h, and washed again with TBST three times. Finally, they were visualized with ECL solution (GE Health Care, Buckinghamshire, UK), and the protein levels were analyzed using Image J software (National Institutes of Health, USA).
6
Proteomic Sample Preparation Protocol
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Dithiothreitol (DTT), iodoacetamide
(IAA), and HEPES were purchased from Sigma-Aldrich Chemie, Steinheim,
Germany, and urea and NH4HCO3 from Acros Organics,
Geel, Belgium. Trypsin, protease inhibitor, dimethyl sulfoxide, Dulbecco’s
Modified Eagle Medium, fetal bovine serum albumin, and the Coomassie
Plus (Bradford) Assay Kit were from Thermo Scientific, Waltham, MA.
Tris(hydroxymethyl)aminomethane hydrochloride (Tris), LC–MS
grade water, acetonitrile, and formic acid were obtained from Fisher
Scientific, Geel, Belgium. RapiGest SF Surfactant was purchased from
Waters Corporation, Milford, MA.
(IAA), and HEPES were purchased from Sigma-Aldrich Chemie, Steinheim,
Germany, and urea and NH4HCO3 from Acros Organics,
Geel, Belgium. Trypsin, protease inhibitor, dimethyl sulfoxide, Dulbecco’s
Modified Eagle Medium, fetal bovine serum albumin, and the Coomassie
Plus (Bradford) Assay Kit were from Thermo Scientific, Waltham, MA.
Tris(hydroxymethyl)aminomethane hydrochloride (Tris), LC–MS
grade water, acetonitrile, and formic acid were obtained from Fisher
Scientific, Geel, Belgium. RapiGest SF Surfactant was purchased from
Waters Corporation, Milford, MA.
7
Peanut Protein Extraction and Quantification
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Peanut flour and 1 X extraction buffer were mixed at a ratio of 1:20 and incubated at 62°C for 10 min with shaking at 2 min intervals. The aqueous peanut protein extract was centrifuged at 3000 x g for 10 min with saving of the supernatant. The protein concentration was determined using the Coomassie Plus™ (Bradford) Assay Kit (Thermo Scientific, Waltham, MA, USA).
8
Western Blot Analysis of SCN9A Protein
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A431 or HEK293 cells were lysed in RIPA buffer (Sigma-Aldrich, #R0278) with protease inhibitors (Sigma-Aldrich, #P8340). Protein concentrations were measured using the Coomassie Plus Bradford assay kit (Thermo Scientific, #23236). For Western blot, 15 µg of whole cell lysates were mixed with loading buffer and boiled for 5 min, and then were run in 4–15% Mini-PROTEAN TGX polyacrylamide gel (Bio-Rad, #456-1083). After transferring, the nitrocellulose membranes were first blotted with 50 mM Tris, 150 mM NaCl and 0.1% Tween-20 (TBS-T) with 5% nonfat milk for 30 min at room temperature. The membranes were then incubated with either anti-SCN9A monoclonal antibody (1:500 dilution in TBS-T, Millipore, #MABN41) or anti-actin monoclonal antibody (1:5,000 dilution in TBS-T, Millipore, #MAB1501) at 4°C overnight. The membranes were then washed with TBS-T three times, each time for 10 min. Subsequently, the membranes were incubated with goat anti-mouse IgG (H+L) secondary antibody (1:10,000 dilution in TBS-T, Thermo Scientific, #31430) at room temperature for 45 min. The membranes were then washed with TBS-T four times, each time for 10 min. The membranes subsequently were incubated with ECL Western blotting substrates (Thermo Scientific, #32106) and then exposed to X-ray films (Sigma-Aldrich, #Z370398).
9
Preparation and Analysis of MEF Cells
10
Glutathione Quantification in Liver Tissue
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Total liver glutathione (GSH) and oxidized glutathione (GSSG) concentrations were determined using a Glutathione Colorimetric Detection Kit (EIAGSHC, Invitrogen). Twenty mg of frozen liver tissue was homogenized with a rod homogenizer (Polytron® PT 2500 E) in ice-cold 1 x phosphate-buffered saline (PBS) and immediately centrifuged (14,000 rpm, 10 min, 4 °C). After protein quantification using the Coomassie Plus (Bradford) Assay Kit (Thermo Scientific), the supernatant was deproteinized with 5% 5-sulfo-salicylic acid dihydrate solution (SSA). For GSSG determination, free GSH and other thiols in the samples were blocked with 2-vinylpyridine (2VP). After adding colorimetric detection reagent and reaction mixture provided with the kit, colorimetric reaction was detected at a wavelength of 405 nm using a Tecan Infinite M 200 pro plate reader. Free glutathione concentration was calculated by subtracting GSSG from GSH. Glutathione levels were normalized for protein content and expressed as μmol/g protein.
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