Lysates from MDA-MB-231 cells were washed once with ice cold PBS (1X), followed by lysis in buffer containing 25 mM Tris [pH 7.5], 0.25% sodium deoxycholate, 1% Triton X-100, supplemented with protease (Roche) and phosphatase inhibitors (10 mM NaF, 1 mM Na3VO4, and 20 mM β-glycerophosphate), unless otherwise noted. Lysates were spun at 15,000 × g for 10 min at 4°C and supernatant was evaluated for protein content using a Pierce BCA Protein Assay (Thermo Fisher Scientific). 25 to 50 μg of protein was loaded onto 8% SDS-PAGE gels or 4–15% Mini-PROTEAN TGX precast gradient gels (Bio-Rad), transferred to nitrocellulose membranes, and probed with the specified antibodies overnight at 4°C in 1X TBS-Tween containing 5% non-fat milk. Immunoblots were visualized using a Bio-Rad ChemiDoc imaging instrument and subsequently processed and quantified using the accompanying Bio-Rad Image Lab software.
Mini protean tgx precast gradient gel
Manufactured by Bio-Rad
Sourced in United States
The Mini-PROTEAN TGX precast gradient gels are a type of lab equipment used for polyacrylamide gel electrophoresis (PAGE) to separate and analyze proteins. They provide a pre-cast, ready-to-use solution for protein separation and analysis.
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Lab products found in correlation
Ecl plus, by Thermo Fisher Scientific (6 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Femto chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail 5, by Merck Group (2 mentions)
Hrp linked goat anti actin, by Santa Cruz Biotechnology (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Las 3000 analyzer, by GE Healthcare (2 mentions)
Chemidoc imaging instrument, by Bio-Rad (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Rabbit anti phospho mtor, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Agilent 1200 series nanoflow system, by Agilent Technologies (1 mentions)
Ltq orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Simplyblue safestain, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Promega (1 mentions)
Butylated hydroxyanisole bha, by Merck Group (1 mentions)
Anti actin antibody, by Thermo Fisher Scientific (1 mentions)
Anti nrf2 antibody, by Active Motif (1 mentions)
Horseradish peroxidase conjugated species specific secondary antibodies, by GE Healthcare (1 mentions)
10 protocols using mini protean tgx precast gradient gel
1
Protein Extraction and Immunoblotting from MDA-MB-231 Cells
2
Phosphorylation Analysis of mTOR and MERTK
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Cultured cells were lysed in buffer containing NaF, Na4P2O7, Na3VO4, and protease inhibitor cocktail V (EMD Millipore, Temecula, CA) to preserve protein phosphorylation. Proteins were resolved on Mini‐PROTEAN TGX precast gradient gels (BioRad, Berkeley, CA), transferred to polyvinylidene fluoride membranes, and incubated with rabbit anti‐phospho‐mTOR (monoclonal antibody [mAb]; Cell Signaling Technology, Danvers, MA), anti‐mTOR (mAb; Cell Signaling Technology), anti‐phospho‐MERTK (mer receptor tyrosine kinase; polyclonal antibody [pAb]; FabGennix, Frisco, TX), or anti‐MERTK (pAb; FabGennix). The blottings were then incubated with horseradish peroxidase (HRP)‐linked anti‐rabbit immunoglobulin G (pAb; Cell Signaling Technology) and HRP‐linked goat anti‐Actin (Santa Cruz Biotechnology, Santa Cruz, CA) and visualized with an 80/20 mix of SuperSignal West Pico Chemiluminescent Substrate and Femto Chemiluminescent Substrate (Thermo Scientific, Rochester, NY). Densitometry was performed using ImageJ analysis software (National Institutes of Health, Bethesda, MD).
3
Protein Extraction and Immunoblot Analysis
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The cells were lysed in buffer containing 10 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, complete protease inhibitor cocktail, and PhoStop EASYpack (Roche). After centrifugation at 15,000 rpm for 20 min at 4 °C, the supernatants were recovered and total protein concentrations were measured using a bicinchoninic acid assay (Pierce BCA Protein Assay Kit; Thermo Scientific). For immunoblot analysis, extracted proteins were separated on 4–15% Mini-PROTEAN TGX precast gradient gels (Bio-Rad) and transferred onto nitrocellulose membranes. The membranes were blocked with 2% nonfat dry milk powder in Tris-buffered saline plus 0.05% Tween and incubated overnight at 4 °C with an anti-MYLK3 antibody (1:500; Novus Biologicals, Littleton, CO, USA; cat# NBP1–86648) recognizing residues 27−144 of human cMLCK and an anti-β-actin antibody (1:5000; Sigma cat# A5441) as a loading control. Primary antibodies were detected with horseradish peroxidase (HRP)-conjugated species-specific secondary antibodies (GE Healthcare) and ECL plus (Thermo Scientific) using a LAS 3000 analyzer (GE Healthcare). Human cardiac tissue and non-transfected HEK293T cells were used as controls. Immunoblot band intensities were measured using ImageJ software (National Institutes of Health).
4
Quantitative Proteomic Analysis of Reversed CB Samples
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Reversed CB samples were separated in mini-protean TGX pre-cast gradient gels (BioRad) and stained with SimplyBlue SafeStain (Life Technologies). Gel pieces were excised from the sample lanes, followed by in-gel digestion with trypsin (Promega) and extraction of the peptides. The peptides were analyzed using LC-MS/MS with an Agilent 1200 series nanoflow system (Agilent Technologies) connected to a LTQ Orbitrap mass-spectrometer (Thermo Electron) equipped with a nanoelectrospray ion source (Proxeon, Odense). The data were analyzed using the Mascot search engine and the NCBI MOUSE database. For the approximate quantitation, the exponentially modified Protein Abundance Index (emPAI) was used.
5
NRF2 Activation in Mouse Heart Tissue
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Heart tissues were homogenized and lysed with RIPA buffer containing 10 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate with protease and phosphatase inhibitor cocktails. Lysates were centrifuged at 20,000 g for 40 min at 4 °C and the supernatants were recovered. Total protein concentrations in the supernatants were measured using a bicinchoninic acid assay (Pierce BCA Protein Assay Kit; Thermo Scientific). For immunoblot analysis, extracted protein samples were separated on 7.5% Mini-PROTEAN TGX precast gradient gels (Bio-Rad) and transferred onto nitrocellulose membranes. The membranes were blocked with 5% FBS in Tris-buffered saline plus 0.05% Tween and incubated overnight at 4 °C with an anti-NRF2 antibody (1:1000; Active Motif) and an anti-actin antibody (1:5000; Thermo Fisher Scientific) as a loading control. Primary antibodies were detected with horseradish peroxidase-conjugated species-specific secondary antibodies (GE Healthcare) and ECL plus (Thermo Fisher Scientific) using a LAS 3000 analyzer (GE Healthcare). Immunoblot band intensities were measured using NIH ImageJ software63 (link). Butylated hydroxyanisole (BHA; Sigma-Aldrich) was administered intraperitoneally to male mice at 8 weeks of age at a dose of 350 mg/kg in corn oil. Uncropped scans of blots are shown in Supplementary Fig. 14a, b, d .
6
Phosphorylation of mTOR and MERTK
7
Western Blot Analysis of ASGR1 and LRP1
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Cells were lysed in RIPA buffer (125 mM Tris HCl, 150 mM NaCl, 0.1% NP-40, 1.0% sodium deoxycholate, 1.0% SDS [pH 7.6]) containing ROCHE protease cocktail phosphatase inhibitors (Sigma Aldrich 4693159001) at the manufacturer’s recommended concentrations. Lysates were loaded onto 10- or 15-well 4%–20% Mini-PROTEAN TGX Precast gradient gels (Bio-Rad, product number 456-1096). The proteins were transferred to a PVDF membrane using the Bio-Rad wet transfer apparatus for 30 min at 90 V or the iblot system from Invitrogen according to the manufacturer’s protocol. PDVF membranes were incubated for 1 hr in odyssey blocking buffer (LI-COR Biosciences cat# 927-40000). The primary antibodies acquired from Abcam, anti-ASGR1 (ab49355) and anti-LRP1 (ab28320), were used at the recommended concentrations. PVDF membranes were then imaged using the Li-Cor Odyssey.50 (link) Infrared detection quantitated each band on an individual pixel basis using western analysis tools in the Image Studio program.50 (link)
8
Quantitative Western Blot Analysis
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Samples were supplemented with 4× Laemmli buffer (catalog no. 1610747; Bio-Rad Laboratories) containing 355 mM 2-mercaptoethanol and denatured for 5 min at 95 °C. Proteins were separated by SDS–gel electrophoresis using 4–20% Mini-PROTEAN TGX precast gradient gels (catalog no. 4561094; Bio-Rad Laboratories) at 100 V for 1.5 h in 1× Tris/glycine/SDS buffer (catalog no. 161–0772; Bio-Rad Laboratories). Wet blotting (25 mM Tris, 192 mM glycine, 20% v/v methanol, pH 8.3) was performed at 100 V for 90 min using methanol-activated polyvinylidene difluoride membranes. Membranes were blocked for 1 h at room temperature in PBS-T (PBS with 0.2% Tween) containing 5% nonfat dry milk powder (NFDM, catalog no. A0830; AppliChem). Primary antibodies (anti-mβ1, directed against the β1 subunit of the sGC44 (link) diluted 1:1,000 and GAPDH, catalog no. 8884S; Cell Signaling Technology, 1:200,000 in 2.5% NFDM-PBS-T) were incubated at 4 °C overnight, followed by a 1-h incubation at room temperature with anti-rabbit HRP-conjugated secondary antibody (1:100,000 in 2.5% NFDM-PBS-T, catalog no. 7074; Cell Signaling Technology). For signal detection, membranes were developed with SuperSignal West Dura Extended Duration Substrate (catalog no. 34075; Thermo Fisher Scientific) according to the manufactureŕs recommendations and signal intensities were detected using an ImageQuant 800 imager (GE Healthcare).
9
Western Blot Analysis of C3aR and C5aR
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50 μg of whole cell lysate in 1x PBS were separated on a 4–20% gradient Mini-PROTEAN TGX precast gradient gel (Bio-Rad) under reducing conditions and transferred to a 0.2 μm Immun-Blot PVDF membrane (Bio-Rad, Hercules, CA, United States). After washing, the membrane was blocked for 1 h with 10% normal donkey serum (NDS) in PBS, followed by probing with goat anti-rat GAPDH (R&D Systems, Minneapolis, MN, United States), followed by a 1:5000 donkey anti-goat IRDye 680 secondary antibody. The membrane was blocked with 10% normal goat serum (NGS) in PBS, followed by probing with rabbit anti-C3aR (Bioss Inc., Woburn, MA, United States) or Mouse anti-C5aR (Hycult Biotech., Plymouth Meeting, PA, United States), followed by a 1:5000 goat anti-rabbit IRDye 800 secondary antibody. The membrane was imaged using a LICOR Clx Odyssey imaging system using both 680 and 800 nm fluorescent channels.
10
Western Blot Protein Detection Protocol
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Purified SHIP1, YopH, and c-Src were resolved on a 4 to 12% Mini-PROTEAN TGX precast gradient gel (Bio-Rad; catalog no.: 4561094) at 200 V for 30 min. Proteins resolved by SDS-PAGE were transferred to nitrocellulose membranes (Bio-Rad; catalog no.: 1704271) using cold 1× transfer buffer (40 ml 5× transfer buffer [Bio-Rad; catalog no.: 1704271], 40 ml ethanol, and 120 ml Milli-Q water). Protein transfer to nitrocellulose membrane was accomplished using the 7 min mixed molecular weight protocol on the Bio-Rad Trans-Blot Turbo Transfer System (Bio-Rad; catalog no.: 1704150). Blots were washed with Milli-Q water and blocked for 1 h with intercept blocking buffer (LI-COR; catalog no.: 927-70001) at 23 °C. The nitrocellulose membrane was then incubated with 1:250 Alexa Fluor 546–labeled phosphotyrosine primary antibody (PY20) (Santa Cruz Biotechnology; catalog no.: sc-508-AF546) diluted in intercept blocking buffer and rocked overnight at 4 °C in the dark. Before visualization, the nitrocellulose membrane was washed four times for 5 min with 1× PBS plus 0.1% Tween-20. The Western blot was visualized using an Amersham Typhoon gel scanner. Data are shown in Fig. S4 D.
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