For preparation of cell extracts, cells were trypsinized off the plate, washed in PBS, and lysed in lysis buffer (50 mM Tris (pH 7.6), 150 mM NaCI, 1% Triton X‐100, and protease inhibitors) for 30 min at 4°C and centrifuged at 15,000 g for 15 min. The protein concentration of the resulting supernatants was determined with the Bradford solution (Bio‐Rad Laboratories, CA, USA). For immunoblotting, equivalent amount of total protein for each cell extract were first resolved on SDS–PAGE gels, transferred onto nitrocellulose membranes, blocked in TBS‐T (Tris‐buffered saline with 0.1% Tween‐20) with 5% milk, blotted with primary antibodies overnight at 4°C and secondary antibodies for 1 h at room temperature. Visualization of the blots was carried out with the LI‐COR Odyssey® Infrared Imaging System and software at 169 μm (LI‐COR Biosciences).
Bradford solution
Manufactured by Bio-Rad
Sourced in United States, Germany
The Bradford solution is a colorimetric assay used for the quantitative determination of protein concentration in a sample. It is a simple, quick, and sensitive method that utilizes the binding of the dye Coomassie Brilliant Blue G-250 to proteins, resulting in a color change that can be measured spectrophotometrically.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (3 mentions)
Bromophenol blue, by Bio-Rad (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Immobilon p pvdf membrane, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Polyvinylidene fluoride pvdf membrane, by Bio-Rad (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
K562 nk cell lines, by Thermo Fisher Scientific (2 mentions)
Pefabloc sc plus, by Merck Group (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Actin, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Vitrobot, by Thermo Fisher Scientific (2 mentions)
Lysis buffer 17, by R&D Systems (1 mentions)
To peroxidase, by Merck Group (1 mentions)
P mapk14, by Cell Signaling Technology (1 mentions)
Eif2ak3, by Cell Signaling Technology (1 mentions)
Chemidoc molecular imager, by Bio-Rad (1 mentions)
37 protocols using bradford solution
1
Protein Isolation and Western Blot Analysis
2
Analyzing Viscum-Induced Cell Signaling
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TC-71 and MHH-ES-1 cells were incubated with viscum, TT or viscumTT in increasing concentrations for 24 h. The cells were washed twice with phosphate-buffered saline and incubated in Lysis Buffer 17 (R&D systems, Minneapolis, MN) containing protease inhibitors (complete Protease Inhibitor Cocktail Tablets, Roche Diagnostics GmbH) to obtain cell lysates. Protein concentration was determined using Bradford solution (Bio-Rad, Munich, Germany). TC-71 and MHH-ES-1 cell lysates (30 μg protein/lane) were separated on SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad system) and blocked with 5% non-fat milk in 50 mM Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h at room temperature. Blots were incubated overnight at 4 °C in TBST containing 5% BSA and primary antibody, washed thrice in TBST and incubated 1 h with HRP-conjugated secondary antibodies (anti-rabbit and anti-mouse, Bio-Rad) then visualized by ECL (Thermo Scientific) on a Molecular Imager ChemiDoc (Bio-Rad). Primary antibodies were directed against p-MAPK14 (Thr180/Tyr182, #9211 Cell Signaling Technology, Danvers, MA, USA), LC3B (#2775 Cell Signaling Technology), EIF2AK3 (#3192, Cell Signaling Technology), p-MAPK8 (sc-6254, Santa Cruz biotechnology, Dallas, TX, USA), HSPA5 (#G8918, Sigma-Aldrich) and ß-actin conjugated directly to peroxidase (#A3854, Sigma-Aldrich).
3
Evaluation of Plumbagin's Antioxidant Potential
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Plumbagin was obtained from the LKT Laboratories (St. Paul, Minnesota, USA). Bradford solution was a product of Bio-Rad (Hercules, CA, USA). Eosin Y 1% aqueous solution and Mayer’s hematoxylin were from Bio Optica (Milan, Italy). Xylene and Permount® were bought from Fisher Scientific (Loughborough, UK). Alanine transaminase (ALT) and aspartate transaminase (AST), bovine serum albumin, β-nicotinamide adenine dinucleotide phosphate reduced form (NADPH), xanthine oxidase, nitroblue tetrazolium (NBT), 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB), SOD, CAT, GSH reductase, malondialdehyde (MDA), thiobarbituric acid (TBA), and 4-vinylpyridine (4-VP) were supplied by Sigma-Aldrich Chemical (St. Louis, Missouri, USA). Ammonium molybdate and hydrogen peroxide (H2O2) were purchased from Ajax Finechem (Melbourne, Australia). Trichloroacetic acid (TCA) was a product of Loba Chemie (Mumbai, India). All other laboratory chemicals were of the highest purity from commercial suppliers.
4
Western Blot Analysis of Protein Expression
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All lysates were prepared in 2 × Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% [v/v] glycerol, 2% [w/v] SDS, 5% [v/v] β-mercaptoethanol, and 0.01% [w/v] bromophenol blue) (Bio-Rad, Hercules, CA). All cellular proteins were quantified using Bradford solution (Bio-Rad) according to the manufacturer’s instructions. The samples were then separated by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad). After blocking with 4% (w/v) skim milk in TBST (25 mM Tris, 140 mM NaCl, and 0.05% [v/v] Tween® 20), the membranes were incubated overnight with specific primary antibodies. Anti-polyglutamylated-tubulin antibody (AG-20B-0020, 1:1000) was purchased from Adipogene (San Diego, CA); anti-GLI2 antibody (18989-1-AP, 1:1000) was purchased from Proteintech (Chicago, IL); anti-actin antibody (MAB1501, 1:10,000) was obtained from Millipore (Temecula, CA); and anti-αPEP7 (tyrosinase) antibody (1:1000) and anti-αPEP1 (TRP1) (1:1000) antibodies were kindly donated by V. J. Hearing (NIH, Bethesda, MD). For protein detection, the membranes were incubated with HRP-conjugated secondary antibodies and the signals were detected with SuperSignal® West Dura HRP Detection Kit (Pierce, Rockford, IL).
5
Cell Lysis and Protein Extraction Protocol
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Cells were lysed on ice in RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol, 1% NP40, 0.5% NaDOC, 0.1% SDS, 2 mM MgCl2, 2 mM CaCl2, 1 mM DTT, 1 mM NaF, 2 mM Na3VO4 and 1× protease inhibitor cocktail (Sigma-Aldrich)) for 20 min, centrifuged and protein concentrations were determined via Bio-Rad Bradford solution according to the manufacturer’s instructions. Proteins were mixed with SDS-PAGE loading buffer (10% glycerol, 2% SDS, 65 mM Tris, 1 mg/100 ml bromophenol blue, 1% β-mercaptoethanol) and equally loaded on a SDS polyacrylamide gel. After size fractionation, proteins were transferred on an Immobilon-P PVDF membrane (Millipore) using an electrophoretic transfer cell (Bio-Rad) and blocked with 5% skim milk powder dissolved in TBS/0.05% Tween for 1 h. The membrane was incubated with primary antibodies at 4 °C overnight or at room temperature for 1 h. HRP-conjugated secondary antibodies were used to visualize specific proteins on a Fusion Fx7 chemoluminescence reader (Vilber Lourmat). Uncropped immuno blot scans from main blots are displayed in Supplementary Fig. 8 .
6
Reagents and Antibodies for Cell Viability Assays
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The following reagents and antibodies were used in the present study: Dulbecco's modified Eagle's medium-nutrient mixture F12 (DMEM-F12; Thermo Fisher Scientific, Inc., Waltham, MA, USA), fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), accutase solution (Merck KGaA, Darmstadt, Germany), alamarBlue Cell Viability reagent (Thermo Fisher Scientific, Inc.), radioimmunoprecipitation assay (RIPA) lysis buffer (Merck KGaA), phosphatase inhibitor (Merck KGaA), protease inhibitor (Merck KGaA), Bradford solution (Bio-Rad Laboratories, Inc., Hercules, CA, USA), bovine serum albumin (BSA) standard solution (New England BioLabs, Inc., Ipswich, MA, USA), PageRuler plus prestained protein ladder (Thermo Fisher Scientific, Inc.), iScript Reverse Transcription SuperMix (Bio-Rad Laboratories, Inc.), Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis kit (Thermo Fisher Scientific, Inc.).
7
Western Blot Analysis of Cellular Proteins
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All lysates were prepared in 2 × Laemmli sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% [v/v] glycerol, 2% [w/v] sodium dodecyl sulphate [SDS], 5% [v/v] β-mercaptoethanol, and 0.01% [w/v] bromophenol blue, Bio-Rad, Hercules, CA, USA). All cellular proteins were quantified using the Bradford solution (Bio-Rad) according to the manufacturer’s instructions. The samples were then separated using SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). After blocking with 4% (w/v) skim milk in Tris-buffered saline plus Tween (TBST; 25 mM Tris, 140 mM sodium chloride [NaCl], and 0.05% [v/v] Tween® 20), the membranes were incubated overnight with the following specific primary antibodies: HA (1:3000, Santa Cruz), SPRR3 (1:3000, Proteintech), Actin (1:10000, EMD Millipore), GAPDH (1:10000, Cell Signaling Technology), phosphor-c-Jun (1:1000 Cell Signaling Technology) and c-Jun (1:3000 Cell Signaling Technology). For protein detection, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and the signals were detected using SuperSignal® West Dura HRP detection kit (Pierce, Rockford, IL, USA).
8
Western Blot Analysis of Luteinized Granulosa Cells
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The cultured luteinized granulosa cells were harvested suspended in cell lysis buffer composed of 62.5 mM Tris–HCl, pH 6.8, 6 M urea, 10% (v/v) glycerol, 2% (w/v) SDS, 0.00125% (w/v) bromophenol blue and freshly added 5% (v/v) β-mercaptoethanol and subjected to sonication on ice. Total protein content was quantified using Bradford assay (Biorad Bradford Solution, USA). 20 µg protein was loaded on 10% SDS–polyacrylamide gel electrophoresis under reducing conditions, along with pre-stained molecular weight markers. The separated proteins were electrophoretically transferred onto a nitrocellulose membrane and incubated overnight at 4 °C with appropriate antibody dilution (supplementary file for table ). The samples were then incubated for 1 h at room temperature with a horseradish peroxidase-conjugated anti-rabbit or anti-goat or anti-mouse IgG and analyzed by Alliance 4.7 UVI Tec chemidoc.
9
Protein Extraction from Cells and Tissues
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For protein isolation, the cells were trypsinized and washed twice with ice-cold PBS. After centrifugation at 240×g and supernatant removal, the cell pellet was resuspended in 50 L of RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 1 mM Na3VO4, 10 mM NaF, 0.1 mM PMSF, 1% NP-40, 1X Roche Complete Protease Inhibitor Cocktail; Roche) and incubated for 30 minutes on ice. The lysate was then sonicated (30-second on/off cycles; Bioruptor Plus; Diagenode) and centrifuged at 15,700×g for 15 minutes at 4°C, followed by transfer to a fresh Eppendorf tube and storage at −80°C. A Bradford assay was used to measure the protein concentration as follows: 1 µL of protein extract was mixed with 999 µL of a 1∶5 dilution of Bradford solution (Bio-Rad) in water. The absorbance at 595 nm was determined with a spectrophotometer (Eppendorf BioPhotometer; Eppendorf). For protein isolation from mouse tissues, the tissues were previously snap-frozen in liquid nitrogen and stored at −80°C. A single tissue piece was minced with a sharp scalpel, followed by the addition of RIPA buffer. The tissue was subsequently homogenized with a tissue homogenizer (Polytron PT 2500E). The tissue lysate was incubated for 30 minutes on ice, followed by the above-described procedure.
10
Western Blot Protein Extraction and Analysis
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Cell extracts were prepared by lysing cells in 50 mM Tris (pH 7.6), 150 mM NaCI, 1% Triton X-100 and protease inhibitors for 30 min at 4 °C followed by centrifugation at 14,000 rpm for 15 min. The protein concentration of lysates was determined with the Bradford solution (Bio-Rad Laboratories, CA, USA) using bovine serum albumin as a standard. For western blotting, cell extracts were resolved on 10% acrylamide sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using the Mini-Protean system (Bio-Rad Laboratories, CA, USA) followed by transfer onto nitrocellulose membranes using the Mini Transblot wet transfer system (Bio-Rad Laboratories, CA, USA). Membranes were incubated in blocking solution (PBS+5% milk, 0.1%Tween-20) for 1 h at room temperature, incubated with primary antibodies overnight at 4 °C and washed three times with PBS+0.1%Tween-20 (PBST). After secondary antibody incubation for 1 h at room temperature and washing three times with PBST, the blots were scanned using the LI-COR Odyssey® scanner and software (LI-COR Biosciences) at 169 µm.
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