Samples were electrophoresed at 100 V for 1 h on a 16% (ubiquitin) or 10% (β-galactosidase) polyacrylamide gel in Tris-Glycine-SDS buffer using a Mini-PROTEAN 3 (Bio-Rad) apparatus. The gel was washed with Tris-Glycine buffer (Thermo Fisher) containing 20% methanol and transferred to a 0.2 μm nitrocellulose membrane (Bio-Rad) in Tris-Glycine buffer containing 20% methanol using a Mini-PROTEAN 3 (Bio-Rad) apparatus. The membrane was blocked for 2 h at room temperature with 1% bovine serum albumin in Tris-buffered saline containing 0.05% Tween 20 (BSA/TBS-T) and incubated overnight at 4 °C with the primary antibody, HRP-conjugated mouse monoclonal IgG1 [Ub Antibody (P4D1), sc-8017] diluted 1:500 with a BSA/ TBS-T mixture. The membrane was washed with TBS. Western blots were developed by incubating the membrane in Super-Signal West Pico Chemiluminescent Substrate (Thermo Scientific) for 5 min at room temperature, and the resulting chemiluminescence was imaged by using a Chem-iDoc XRS (Bio-Rad) instrument equipped with Quantity One software.
Mini protean 3
Manufactured by Bio-Rad
Sourced in United States, United Kingdom, Italy
The Mini-Protean III is a compact vertical electrophoresis system designed for analytical and preparative protein separations. It features a simple, intuitive setup and accommodates a variety of gel formats and sample sizes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (8 mentions)
Ab6721, by Abcam (6 mentions)
Sc 19648, by Santa Cruz Biotechnology (6 mentions)
Bca kit, by Solarbio (6 mentions)
Ab131097, by Abcam (6 mentions)
Ab32391, by Abcam (6 mentions)
Ab16051, by Abcam (6 mentions)
Ab16663, by Abcam (6 mentions)
Sc 2354, by Santa Cruz Biotechnology (6 mentions)
Lysozyme solution, by Thermo Fisher Scientific (5 mentions)
Ab8227, by Abcam (5 mentions)
Chemidoc xr, by Bio-Rad (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Hybond, by Cytiva (3 mentions)
Ecl western blotting substrate kit, by Thermo Fisher Scientific (2 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
Immobilon p, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Gs 800 calibrated densitometer, by Bio-Rad (2 mentions)
Thermomixer comfort, by Eppendorf (2 mentions)
79 protocols using mini protean 3
1
Western Blot Analysis of Ubiquitin and β-Galactosidase
2
SDS-PAGE and native electrophoresis of proteins
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Samples for SDS polyacrylamide gels (PAGE) were denatured for 10 min at 60 °C in a sample lysis buffer containing 50 mM Tris-HCl pH 7.0, 4% (w/v) SDS, 10% (v/v) glycerol and 0.1 M 1,4-dithiothreitol and separated on a 12% polyacrylamide minigels (MiniProtean III, Bio-Rad, Hercules, CA, USA) using the Tricine buffer system [25 (link)]. To achieve better separation, for analysis of HEK293 overexpressing mGPDH-FLAG, midi gel system Hoefer SE 600 (Thermo Fisher) was used.
For native electrophoresis, cells were centrifuged 10 min at 600× g and resulting pellets were resuspended in buffer containing 50 mM NaCl, 2 mM 6-aminohexanoic acid, 50 mM imidazole and 1 mM EDTA, pH 7.0. Proteins were solubilized with digitonin (2 g/g protein) for 10 min on ice and centrifuged for 20 min at 30,000× g to remove cell debris. Ponceau red dye (0.01%) and 10% glycerol were added to the supernatants and the samples were analyzed by high-resolution clear-native electrophoresis 3 (hrCNE, [26 (link)]) using 5–13% polyacrylamide gradient mini gels (MiniProtean III, Bio-Rad).
For two-dimensional (2D) analyses, strips of the first dimension gel (hrCNE) were incubated in 2D buffer containing 1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol for 1 h at room temperature and then resolved in the second dimension on SDS-PAGE as described above.
For native electrophoresis, cells were centrifuged 10 min at 600× g and resulting pellets were resuspended in buffer containing 50 mM NaCl, 2 mM 6-aminohexanoic acid, 50 mM imidazole and 1 mM EDTA, pH 7.0. Proteins were solubilized with digitonin (2 g/g protein) for 10 min on ice and centrifuged for 20 min at 30,000× g to remove cell debris. Ponceau red dye (0.01%) and 10% glycerol were added to the supernatants and the samples were analyzed by high-resolution clear-native electrophoresis 3 (hrCNE, [26 (link)]) using 5–13% polyacrylamide gradient mini gels (MiniProtean III, Bio-Rad).
For two-dimensional (2D) analyses, strips of the first dimension gel (hrCNE) were incubated in 2D buffer containing 1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol for 1 h at room temperature and then resolved in the second dimension on SDS-PAGE as described above.
3
Collagen Type I SDS-PAGE Analysis
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Cell layers were analysed by SDS-PAGE as described elsewhere (Capella-Monsonís et al., 2018 (link)). Briefly, culture media were aspirated, cell layers were washed with PBS and digested with 0.1 mg/ml pepsin solution (porcine gastric mucosa, 3,500–4,200 U/mg) in 0.5 M acetic acid. The cell layers were then scraped, neutralised with 1 M NaOH, denatured at 95°C and resolved under non-reducing conditions using in-house resolving and stacking polyacrylamide gels (5 and 3% respectively) on a Mini-Protean 3 (Bio-Rad Laboratories, United Kingdom) system. Purified collagen type I (Symatese, France) was used as standard. Samples were stained using a SilverQuest™ Silver Staining Kit (Invitrogen, Ireland) according to the manufacturer’s instructions. Densitometric analysis was performed on α1(I), α2(I), β11(I), β12(I) or γ(I) bands, as appropriate, using ImageJ software (NIH, United States).
4
Proteomic Analysis of Thermophilic C. bescii
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Five hundred mL cultures of C. bescii strains (JWCB017, 049, and 054) were grown to mid-log phase at 75 °C, harvested by centrifugation at 6000×g at 4 °C for 15 min and resuspended in Cell-Lytic B cell lysis reagent (Sigma-Aldrich, St. Louis, MO, USA). Cells were lysed by a combination of 4× freeze-thawing and sonication on ice. Protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) with bovine serum albumin as the standard. Cell free extracts (75 µg) were electrophoresed in 4–15 % gradient Mini-Protean TGX gels, which were either stained using Coomassie blue or were transferred to PVDF membranes (ImmobilonTM-P; EMD Millipore, Billerica, MA, USA) using a Bio-Rad Mini-Protean 3 electrophoretic apparatus. The membrane was then probed with His-tag (6xHis) monoclonal antibody (1:5000 dilution; Invitrogen, Grand Island, NY, USA) using the ECL Western Blotting substrate Kit (Thermo Scientific, Waltham, MA, USA) as specified by the manufacturer.
5
Immunoblotting Procedure Modifications
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Immunoblotting procedures were modified from Lee et al. [13 (link), 15 (link)] with a few modifications. Aliquots containing 20 μg of sample homogenates were heated with denaturing buffer at 60°C for 15 min. The samples were separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels. The separated proteins were transferred to 0.45 μm polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA) using a tank transfer system (Mini protean 3; Bio-Rad, Hercules, CA, USA). The membranes were pre-incubated for 2 h in PBS containing 0.2% (v/v) Tween 20 and 5% (w/v) nonfat dried milk to minimize nonspecific binding. Next, the blots were incubated at 4°C overnight with antibodies (Table 2 ). The membranes were then incubated at room temperature for 1 h with secondary antibodies (Table 2 ). Blots were developed using ImmobilonTW Western (Millipore). Signals were obtained using the Chemidoc XRS+ image system (Bio-Rad), and then the data were analyzed with Image Lab software (version 3.0; Bio-Rad). The results were converted to numerical values to compare the relative protein abundance of the immunoreactive bands.
6
Proteomic Analysis by 2D-PAGE and MALDI-TOF/TOF
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Protean IEF cell, IPG strips pH 3–10 (7 cm), IEF focusing tray with lid, SDS-PAGE (SDS-Polyacrylamide gel) electrophoresis cell (model Mini-PROTEAN 3) were from Bio-Rad, following the manufacturer’s instructions. The image of the gel after staining was acquired in a PROPIC II DigiLab Genomic Solutions USA. Protein digestion was done in safe-lock tubes of 1.5 mL from Eppendorf (Hamburg, Germany). A vacuum concentrator centrifuge model UNIVAPO 150 ECH SpeedVac and a vacuum pump model UNIJET II were used for sample drying and sample pre-concentration. A mini incubator from Labnet was used for gel washing, for protein reduction and for protein alkylation steps. The centrifuge MPW-350 and MPW-65R were from MPW Med. Instruments. Absorption spectra of samples were recorded as microliter samples using a NANODROP ND-1000 Spectrophotometer from Thermo Scientific (USA). Protein identification was done in an Ultraflex II MALDI-TOF/TOF instrument from Bruker Daltonics.
7
Quantification of Skin and Cell Proteins
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Total protein from skin tissue and BJ cells was extracted using RIPA buffer (Beyotime Institute of Biotechnology) and then quantified using a Pierce™ BCA Protein assay kit (cat. no. 23225; Thermo Fisher Scientific, Inc.). A total of 40 µg protein/lane was separated via SDS-PAGE (Mini-Protean-3; Bio-Rad Laboratories, Inc.) and transferred to polyvinylidene difluoride membranes (EMD Millipore). The membrane was blocked with 5% skimmed milk powder solution for 1 h and then incubated with the following primary antibodies at 4˚C overnight: Rabbit anti-collagen I antibody (cat. no. ab34710; 1:2,000 dilution; Abcam), rabbit anti-collagen III antibody (cat. no. ab7778; 1:5,000 dilution; Abcam), rabbit anti-MMP-1 antibody (cat. no. ab137332; 1:1,000 dilution; Abcam), rabbit anti-α-SMA antibody (cat. no. YM-H0645; 1:500 dilution; Shanghai Yuan Mu Biotechnology Co., Ltd.) and rabbit anti-β-actin antibody (cat. no. ab8227; 1:2,00 dilution; Abcam). The protein bands were then incubated with secondary antibody goat anti-rabbit IgG (cat. no. ab6721; 1:2,000 dilution; Abcam) at room temperature for 2 h and treated with enhanced chemiluminescence solution (Thermo Fisher Scientific, Inc.). β-actin was used as the internal reference and the gray values of protein bands were quantitatively analyzed by ImageJ software (version 1.46r; National Institutes of Health).
8
One-Dimensional Electrophoresis of Membrane Proteins
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To perform one-dimensional electrophoresis, membrane proteins were solubilized in Laemmli buffer [51 (link)] at a volume ratio of 1:1.30 µg of protein-to-antiphosphotyrosine. Then, the samples were put in a thermomixer at 1400 rpm and 28 °C for 30 min. Next, the samples were stored at 95 °C for 5 min, then separated on 8% polyacrylamide gel under reducing and non-reducing conditions. The electrophoretic run was performed on the Bio-Rad Mini-Protean 3 setup.
9
SDS-PAGE Protein Analysis Protocol
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Electrophoresis experiments were carried out using a Mini-PROTEAN® 3 or a Mini-PROTEAN® Tetra cell electrophoresis unit (Bio-Rad Laboratories, Inc., USA). The protocols (SDS-PAGE) were carried out according to the manufacturer’s instructions. Reagents of electrophoresis grade were obtained from Bio-Rad Laboratories Inc., electrophoresis grade ammonium persulfate was purchased from Sigma Aldrich and 1,2-bis(dimethylamino)ethane was obtained from Merck Millipore. A broad range of molecular weight markers were purchased from Bio-rad Laboratories Inc. (Precision Plus Protein™ Dual Color Standards) and Thermo Scientific (Scientific PageRuler Plus Prestained Protein Ladder). Unless stated otherwise, samples were prepared following the Laemmli protocol [35 (link)]. Dithiothreitol (DTT, Bio-rad Laboratories Inc.) was selected as the reducing agent. DTT was added to a final 1x concentration of 50 mM. Samples were run on 10% SDS-PAGE vertical minigels under both non-reducing/reducing conditions (DTT included). Electrophoretic conditions were 200 V at constant current according to the manufacturer’s instructions. Minigels were stained with Coomassie blue or silver.
10
Western Blot Analysis of Oxidative Stress Markers
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The cells were homogenized, washed with PBS and dissolved in RIPA buffer (Beyotime, Shanghai, China) and the quantitative protein concentration was determined by Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China). The total protein content was quantified and equal amounts of protein were loaded on 8–12% SDS-polyacrylamide gel electrophoresis (Mini-Protean-3, Bio-Rad, Hercules, CA, U.S.A.) and transferred onto a PVDF transfer membrane (Millipore, Massachusetts, U.S.A.). After blocking the membrane, they were incubated with specific primary antibodies, Nrf-2 (1:1000, ab137550), HO-1 (1:1000, ab13243), TGF-β (1:1000, ab190503), p-smad-3 (1:1000, ab193297), smad-3 (1:1000, ab40854), p-IκBα (1:1000, ab133462), IκBα (1:1000, ab32518), p-NF-κBp65 (1:1000, ab86299), NF-κBp65 (1:1000, ab16502), overnight at 4°C followed by further incubation with the secondary antibody at room temperature for 2 h. Visualization of protein bands was detected with ECL chemoluminescence staining detection kit (Bio-Rad) using densitometry of Bandscan 5.0 software that was used for quantifying the density of each protein band. With GAPDH as control, the expression of total protein was normalized.
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