After bear serum incubation, cells were scraped into 200 μl of ice-cold lysis buffer (TRIS-HCl 20 mM, NaCl 138 mM, KCl 2.7 mM, MgCl2 1 mM, Glycerol 5%, NP 40 1%, EDTA 5 mM, Na3VO4 1 mM, NaF 20 mM and DTT 1 mM) supplemented with a protease inhibitor cocktail (Sigma-Aldrich, France). Protein concentration was determined by Bradford quantification. Western blotting was performed, as described previously29 (link), loading 20 μg of total protein on precast gels (Mini-protean TGX Stain-freeTM gel, Biorad, France). After migration, gels were UV exposed for 3 minutes and pictures were taken for further quantification of protein loading. After semi-dry transfer, all membranes were blocked with 4% BSA (Bovine Serum Albumin, Euromedex, Souffelweyersheim, France) before incubation with primary antibodies (see Table S1 ). Corresponding secondary HRP antibodies were used for chemiluminescence revelation (Chemidoc Bio-Rad).
Mini protean tgx stain free gel
Manufactured by Bio-Rad
Sourced in United States, Australia, United Kingdom, Belgium, Germany
The Mini-PROTEAN TGX Stain-Free Gels are precast polyacrylamide gels designed for electrophoretic separation of proteins. The gels feature a stain-free technology that enables direct visualization of proteins without the need for traditional staining methods.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo transfer system, by Bio-Rad (27 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (22 mentions)
Clarity western ecl substrate, by Bio-Rad (18 mentions)
Chemidoc mp imaging system, by Bio-Rad (17 mentions)
Chemidoc touch imaging system, by Bio-Rad (13 mentions)
Nitrocellulose membrane, by Bio-Rad (11 mentions)
Chemidoc mp system, by Bio-Rad (11 mentions)
Chemidoc imaging system, by Bio-Rad (10 mentions)
Trans blot turbo transfer pack, by Bio-Rad (9 mentions)
Trans blot turbo system, by Bio-Rad (8 mentions)
Image lab software, by Bio-Rad (7 mentions)
Laemmli sample buffer, by Bio-Rad (7 mentions)
Pvdf membrane, by Bio-Rad (6 mentions)
Odyssey blocking buffer, by LI COR (6 mentions)
Laemmli buffer, by Bio-Rad (6 mentions)
Protease inhibitor cocktail, by Roche (6 mentions)
Ecl chemiluminescence system, by Thermo Fisher Scientific (6 mentions)
Dc protein assay, by Bio-Rad (6 mentions)
Bca protein assay, by Thermo Fisher Scientific (6 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (5 mentions)
220 protocols using mini protean tgx stain free gel
1
Protein extraction and Western blot analysis
2
Quantifying Wound Angiogenesis and Inflammation
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To measure the CD31 and F4/80 protein levels, mice were sacrificed and wound tissues were harvested 7 days after treatment. Briefly, the harvested wound tissues were homogenized using Multi-beads shocker® and added to the T-PER Reagent (Thermo Fisher Scientific) consisting of proteinase and dephosphorylation inhibitors (Thermo Fisher Scientific). Total protein lysates were obtained after the elimination of debris by filtering with 0.22 μm filters (Millipore). Total protein (10 μg) was separated using a mini-protean TGX Stain-FreeTM gel (BioRad), and then transferred to PVDF membranes using the Trans-Blot® TurboTM transfer system (BioRad). After blocking with the PVDF Blocking Reagent Can Get Signal® (TOYOBO) for 1 hour at room temperature, the membranes were incubated with primary antibodies against CD31, F4/80, or tubulin (Sigma-Aldrich) at 4 °C overnight, followed by the appropriate horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 hour. Expression was visualized by chemiluminescence with a digital luminescent image analyzer (LAS-4000, GE Healthcare).
3
Proteomic Analysis of Basal Bodies
4
Immunoblotting of Dermal Fibroblast Proteins
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Dermal fibroblast cells were lysed in solution containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA (pH 8), 1% NP40 and 2.5% SDS supplemented with protease inhibitors (Halt Protease Inhibitor Cocktail, Thermo Fisher). Protein samples were loaded to a 4–20% Mini-PROTEAN TGX stain-free gel (Bio-Rad), transferred to a PVDF membrane and incubated with respective primary antibodies. The antibodies and the dilutions used in this study were the following: anti-SMC2 (Novus Biologicals, NB100-373), 1:2000; anti-SMC4 (Proteintech, 24758-1-AP), anti-ß-Actin (Cell Signaling, #4970), 1:1000. After incubating the membrane with horseradish peroxidase-conjugated secondary antibodies, the proteins were visualized with enhanced chemiluminescence detection (WesternBright ECL HRP substrate, Advansta) method.
5
SDS-PAGE Protein Sample Preparation
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For SDS–PAGE, protein samples were denatured by boiling them with Pierce Lane Marker Reducing Sample Buffer (Thermo Fisher Scientific) for 10 min at 98 °C. After centrifuging the samples at 17,000 × g, the clear supernatant was loaded on a 4–20% Mini-PROTEAN TGX Stain-Free Gel (Bio-Rad Laboratories) including PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) as a molecular weight reference. The electrophoresis was run for 30 min at a constant voltage of 180 V before staining the gel with GelCode Blue Safe Protein Stain (Thermo Fisher Scientific).
6
Proteomic Analysis of Basal Bodies
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A sample of purified basal bodies was run briefly into a 4-20% Mini-PROTEAN® TGX Stain-Free™ gel (BIO-RAD), which was then stained with InstantBlue™ before the gel section containing the sample was excised and sent for analysis at either the BSRC Mass Spectrometry Facility (University of St Andrews), or the Advanced Proteomics Facility (Department of Biochemistry, University of Oxford).
7
Protein Expression Analysis Protocol
8
Western Blot Analysis of B3GAT1 Protein
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Indicated cell lines were lysed by pelleting 3 × 106 cells, resuspending in 200 µL lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100), and incubating for 10 min at room temperature. Supernatant was combined with Laemmli sample buffer and 5% 2-beta mercaptoethanol and heated at 95 °C for 5 min. Lysate was then loaded onto a 4–20% Mini-PROTEAN TGX Stain-Free Gel (Bio-Rad). After running samples, stain-free gels were imaged on a Bio-Rad ChemiDoc Imaging System, and protein was transferred to a nitrocellulose blotting membrane (GE 10600002) for 60 min at 60 V. Membranes were blocked in 5% nonfat dry milk in PBS + 0.1% Tween-20 (0.1% PBS-T) for 1–2 h at 4 °C, then incubated overnight at 4 °C in anti-B3GAT1 antibody (Sigma AMAB91575, 1:1000) diluted in blocking buffer. Membranes were washed with 0.1% PBS-T, incubated in goat anti-mouse HRP secondary (Invitrogen A16072, 1:5000) for 1 h at room temperature, and washed again with 0.1% PBS-T. To develop blots, membranes were incubated in Clarity Western ECL substrate (Bio-Rad) and exposed to film.
9
Membrane Protein Extraction and Western Blot Analysis
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Membrane protein lysates were harvested from 60-mm dishes following the instructions in the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher, United Kingdom), supplemented with proteasome inhibitor cocktail (Sigma-Aldrich, United Kingdom). Protein lysates were prepared with 2x Laemmli sample buffer (1: 1) (Sigma-Aldrich, United Kingdom) supplemented with β-mercaptoethanol (Sigma-Aldrich, United Kingdom). These were loaded on a 4%–20% Mini-PROTEAN TGX Stain-Free Gel (Bio-Rad, United Kingdom). Gels were transferred onto a nitrocellulose membrane (LI-COR Biosciences, United Kingdom) by wet transfer at 350 mA for 90 min at 4°C. The membrane was blocked in 5% milk in TBS-T at room temperature for 1 h before adding the primary antibody in 2% milk TBS-T for 1 h at room temperature. The membrane was washed three times in TBS-T and incubated with the secondary antibody in 2% milk TBS-T for 1 h at room temperature. After three washes in TBS-T, the membrane was imaged using the LI-COR Odyssey CLx system, and LI-COR Image Studio v5.0 was used to analyze the image. The antibodies used in this study are listed in Supplementary Tables S3, S4 .
10
Western Blot Analysis of MET, CPD, and GAPDH
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Cells were washed with PBS and harvested in RIPA lysis buffer (Thermo Fisher Scientific, 89900) supplemented with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, 87786). Twenty micrograms of cell lysate was denatured in Laemmli SDS-Sample Buffer (Boston BioProducts, BP-110R) and loaded on a precast 4–15% Mini-Protean TGX Stain-Free gel (Bio-Rad). After electrophoresis and transfer, the PVDF membrane (Bio-Rad) was blocked in 5% nonfat milk with TBST (Bio-Rad) at room temperature for 1 h and incubated with primary antibody overnight. MET (3127, 1:1000) antibody was purchased from cell signaling technology. CPD (A305-514A-M, 1:1000) and GAPDH (MA5-15738, 1:5000) antibodies were purchased from Thermo Fisher Scientific. The membranes were then washed in TBST three times, followed by incubation with goat anti-rabbit/Mouse secondary antibodies (Cell Signaling Technology, 1:2000–1:5000) for 1 h at room temperature. After washing with TBST, the membranes were developed by films. All western blots were repeated twice. Image quantification was performed in ImageJ.
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