Cell pellets from 1 L LB culture were suspended with 30 mL of buffer A (20 mM Tris-HCl (pH 7.9), 300 mM NaCl, 20 mM imidazole, 5 mM beta-mercaptoethanol (BME), 1% Tween20) supplemented with a tablet protease inhibitor cocktail (complete, EDTA-Free, Sigma). Cells were lysed by sonication. Soluble fractions were separated by centrifugation (20,000 × g for 40 min at 4 °C). The supernatant was loaded on a pre-equilibrated 1 mL HisTrap column (GE Healthcare, USA) with buffer B (20 mM Tris-HCl (pH 7.9), 300 mM NaCl, 20 mM imidazole) and washed with 10 column volumes (CV) of buffer B and 50 CV of buffer C (20 mM Tris-HCl (pH 7.9), 300 mM NaCl, 50 mM imidazole), and eluted with buffer D (20 mM Tris-HCl (pH 7.9), 300 mM NaCl, 300 mM imidazole). Eluent was injected to HiLoad 26/600 superdex 200 pg (GE Healthcare, USA) column equilibrated with buffer E (20 mM HEPES pH 7.5, 300 mM NaCl, 10% Glycerol, 0.5 mM TCEP). The major fractions were analyzed with 4–20% SDS-PAGE Gel stained with coomassie brilliant blue. Protein concentrations were determined by Bradford protein assay kit II (Bio-Rad, USA) with bovine serum albumin as the standard.
Bradford protein assay kit 2
Manufactured by Bio-Rad
Sourced in United States
The Bradford protein assay kit II is a colorimetric assay used for the quantitation of total protein concentration in a sample. It relies on 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
Complete edta free, by Merck Group (1 mentions)
Hiload 26 600 superdex200 pg, by GE Healthcare (1 mentions)
Model 680 microplate reader, by Bio-Rad (1 mentions)
Citrate synthase assay kit, by Merck Group (1 mentions)
Ultrasonic homogenizer 4710 series, by Cole-Parmer (1 mentions)
Pvdf blotting membrane, by GE Healthcare (1 mentions)
Sc 5275, by Santa Cruz Biotechnology (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Thermo Fisher Scientific (1 mentions)
Sds polyacrylamide gel electrophoresis, by Bio-Rad (1 mentions)
Sc 8059, by Santa Cruz Biotechnology (1 mentions)
Ab22656, by Abcam (1 mentions)
Ab28356, by Abcam (1 mentions)
Gel densitometer, by Bio-Rad (1 mentions)
Ab205719, by Abcam (1 mentions)
Polyvinyl difluoride membrane, by Merck Group (1 mentions)
Xmark microplate spectrophotometer, by Bio-Rad (1 mentions)
6 protocols using bradford protein assay kit 2
1
Purification of His-Tagged Proteins
2
Mitochondrial Respiration and Protein Quantification
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Respiration data from the Seahorse experiments were normalized to citrate synthase activity and protein content. Citrate synthase activity was analyzed using Citrate Synthase Assay Kit (Sigma-Aldrich, St. Louis, US). Cells from all cell lines were resuspended in mitochondrial respiration medium (MiR05) (sucrose 110 mM, HEPES 20 mM, taurine 20 mM, K-lactobionate 60 mM, MgCl2 3 mM, KH2PO4 10 mM, EGTA 0.5 mM, BSA 1 g/l) to a concentration of 3 x 106 cells/mL, then sonicated using Ultrasonic homogenizer 4710 Series (Cole-Parmer Instrument, Vernon Hills, US). Samples were added to a 96-well plate together with 1x Assay buffer, 30 mM acetyl-coenzyme A and 10 mM 5,5’-Dithiobis-2-nitrobenzoic acid. 10 mM oxaloacetic acid was added and citrate synthase measured with BIO-RAD Microplate Reader Model 680 (Bio-Rad, Hercules, US).
Protein contents were analyzed with Bradford Protein Assay Kit II (Bio-Rad, Hercules, US). After each Seahorse experiment, the plates were washed with 1x phosphate-buffered saline and frozen at -80°C. Before assay, the cells were thawed and lysed with Radioimmunoprecipitation Assay lysis buffer. 10 µL of cell lysate was mixed with 190 µl Protein Assay Dye Reagent and transferred to assay plate. Absorbance was recorded using Microplate Reader Model 680 (Bio-Rad, Hercules, US) and compared against Bradford Assay Standards.
Protein contents were analyzed with Bradford Protein Assay Kit II (Bio-Rad, Hercules, US). After each Seahorse experiment, the plates were washed with 1x phosphate-buffered saline and frozen at -80°C. Before assay, the cells were thawed and lysed with Radioimmunoprecipitation Assay lysis buffer. 10 µL of cell lysate was mixed with 190 µl Protein Assay Dye Reagent and transferred to assay plate. Absorbance was recorded using Microplate Reader Model 680 (Bio-Rad, Hercules, US) and compared against Bradford Assay Standards.
3
Protein Extraction and Western Blot Analysis of Extracellular Vesicles
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The EVs (microvesicles or exosomes) were lysed using lysis buffer (50 mmol/L Tris-HCl pH 7.2, 5 mmol/L MgCl2, 50 mmol/L NaCl, 0.25%, 0.1% SDS, and 1% Triton X-100) containing protease inhibitors (2 mmol/L phenyl methyl sulfonyl fluoride, 10 mg/mL aprotinin, and 2 mmol/L Na3VO4, 100 mmol/L NaF). Protein concentration was assessed using the Bradford method (Bradford protein assay kit II, Bio-Rad, Hercules, CA, USA), with BSA used as a standard. EVs extracted proteins (5–10 µg) were resolved by SDS PAGE (Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis) 10% under reducing or non-reducing conditions, and were transferred to PVDF blotting membranes (GE Healthcare, Solingen, Germany) and analyzed using the enhanced chemiluminescence kit for Western blotting detection (Advansta, WesternBright TM ECL, Bering Drive San Jose, CA, USA). CD63 primary monoclonal antibody was used following suppliers’ (dilution, 1:500; sc-5275; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). This antibody recognizes the glycosylated forms of CD63 (30–60 kDa).
4
Protein Expression Analysis by Western Blot
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Cells were dissolved in 2× sodium dodecyl sulfate (SDS) protein sample buffer which contained 100 mM Tris-HCl (pH = 6.8), 200 mM DTT, 4% SDS, 0.4% bromophenol blue, and 20% glycerol. The protein concentration was determined using a Bradford Protein assay kit II (Bio-Rad Laboratories, Hercules, CA, USA). After that, an equal amount of protein sample (25 μg) was separated on 10% SDS-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA) and transferred on polyvinylidene fluoride membranes (Thermo Fisher Scientific). After being blocked by 1% bovine serum albumin for 1 h, the membranes were incubated with the primary antibodies to β-catenin (1 : 1,000, ab22656, Abcam), p-AKT (1 : 500, 66444-1-Ig, Proteintech Group, Chicago, IL, USA), AKT (1 : 800, 60203-2-Ig, Proteintech Group), p-STAT3 (1 : 2,000, sc-8059, Santa Cruz Biotechnology), p-PI3K (1 : 800, ab28356, Abcam), E-cadherin (1 : 800, 33-4000, Thermo Fisher scientific), Vimentin (1 : 1,000, MA5-11883, Thermo Fisher scientific), and GAPDH (1 : 800, 60004-1-Ig, Proteintech Group) at 4°C overnight. After that, the membranes were further incubated with alkaline phosphatase-conjugated goat anti-mouse (1 : 4,000, ab205719, Abcam) at 20°C for 1 h. The protein bands were visualized using the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific), and the image density was analyzed using a gel densitometer (Bio-Rad).
5
Western Blot Analysis of EMT Markers
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Following the aforementioned treatments, cells were washed twice with PBS, harvested and solubilized in 2X sodium dodecyl sulfate (SDS) protein sample buffer containing 100 mM Tris-HCl (pH 6.8), 200 mM DTT, 4 SDS, 0.4% bromophenol blue, and 20% glycerol. Protein was quantified using the Bradford Protein assay kit II (Bio-Rad Laboratories, Inc.). Equal quantities of protein (25 µg) were separated by 10% SDS-polyacrylamide gel electrophoresis. The resolved proteins were then transferred to polyvinyldifluoride membranes (Merck Millipore). The membranes were blocked by incubation with 1% bovine serum albumin in TBS-Tween-20 (TBST; 10 mM Tris-HCl, 150 mM NaCl pH 7.5 containing 0.1% Tween-20) at room temperature for 1 h and were then incubated with primary antibodies against HIF1α (1:500), E-cadherin (1:200), vimentin (1:500), Snail1 (1:200) and β-actin (1:1,000) for 1 h at room temperature. The membranes were washed three times with TBST and were then incubated for 30 min with goat anti-rabbit or goat anti-mouse secondary antibody conjugated to alkaline phosphatase (1:1,000; Santa Cruz Biotechnology, Inc.; cat. nos. sc-2007 and sc-2008). The respective proteins were detected by colorimetric reactions using 5-bromo-4-chloro-3′-indolyphosphate/nitro-blue tetrazolium substrate.
6
Epididymal Tissue Protein Extraction
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The epididymal tissue was homogenized in an appropriate volume of lysis buffer (with freshly added 20 mM of sodium fluoride, 100 mM sodium orthovanadate, and 1% of protease inhibitor cocktail) and allowed to stand 20 min on ice. The homogenates were centrifuged at 14,000× g in the Hettich Mikro 200 R centrifuge for 20 min at 4 °C. After centrifugation, the pellet was discarded. The total protein concentration was quantified using the Bradford Protein Assay Kit II from Bio-Rad (Hercules, CA, USA) according to the manufacturer’s instructions, and the absorbance was measured by the xMark Microplate Spectrophotometer from Bio-Rad (Hercules, CA, USA). The protein concentration was determined using different bovine serum albumin (BSA) concentrations as standards for calibration. Optical densities of samples were determined at 595 nm [7 (link)].
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