Lysates of the Dox titration and receptor-capture experiments were analyzed through western blotting. A bicinchoninic assay (Expedeon, Cambridge, UK) was used according to the manufacturer’s protocol to determine and equalize the protein concentrations of the samples. SDS-PAGE sample buffer was added to the samples, and they were heated to 65°C for 5 minutes. Ten to 20 μg of protein per sample was loaded into wells of 4%–12% BisTris precast NuPAGE or BOLT gels (Thermo Fisher Scientific) and subjected to SDS-PAGE in NuPAGE or BOLT MOPS SDS running buffer (Thermo Fisher Scientific). The proteins were then electrophoretically transferred to a nitrocellulose membrane, which was blocked in PBS blocking buffer (LI-COR) and subsequently probed for HA-NK1-eGFP, HA-NK1-6xHis, tubulin, and/or biotin using the appropriate primary and secondary antibodies, or streptavidin diluted in PBS blocking buffer (LI-COR) supplemented with 0.2% Tween 20. An Odyssey Scanner (LI-COR) was used to image the membranes.
Bolt gel
Manufactured by Thermo Fisher Scientific
Sourced in United States
Bolt Gels are a type of electrophoresis gel used for the separation and analysis of proteins and nucleic acids. They provide a consistent and reliable platform for the resolution of complex samples. The gels are designed to offer high-quality results with a focus on performance and reproducibility.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Thermo Fisher Scientific (3 mentions)
Gtu 88, by Merck Group (2 mentions)
Ab219649, by Abcam (2 mentions)
F3165, by OriGene (2 mentions)
Ta180093, by OriGene (2 mentions)
Jbw301 05 636, by Merck Group (2 mentions)
Hpa040067, by Abcam (2 mentions)
Re blot plus strong solution, by Merck Group (2 mentions)
Clarity ecl, by Bio-Rad (2 mentions)
Phosstop phosphatase inhibitor, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Hydrogen peroxide 30, by Merck Group (2 mentions)
Tissuelyser, by Qiagen (2 mentions)
Bca assay, by Thermo Fisher Scientific (2 mentions)
Nupage, by Thermo Fisher Scientific (2 mentions)
Bolt mops sds running buffer, by Thermo Fisher Scientific (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
Pbs blocking buffer, by LI COR (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
22 protocols using bolt gel
1
Western Blot Analysis of Receptor Proteins
2
Whole-Cell Extraction Western Blot
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Whole-cell extracts (WCE) were prepared by lysing cells in two volumes of Lysis buffer (50 mmol/L Tris pH 7.4, 150 mmol/L NaCl, 5 mmol/L EDTA, 50 mmol/L HEPES, 1% v/v Triton-X100, 0.1% v/v SDS). Thirty to 60 μg of WCE was resolved on 4%–12% Bolt Gels (Thermo Fisher Scientific). Protein was transferred to nitrocellulose membrane (Thermo Fisher Scientific) and blotted for SF3B1 (Bethyl Laboratories: A300–996A; RRID:AB_805834)), Flag M2 (Sigma-Aldrich: F3165; RRID:AB_439685), mRFP (Origene:TA180093; RRID:AB_2622287) and γ-tubulin (GTU-88:Sigma Aldrich; RRID:AB_532292), ATM (2C1:Abcam; RRID:AB_368161), BRCA2 (OP95:Millipore; RRID: AB_2067762), ATR (N19:SCBT; RRID:AB_630893), Mre11 (4895:Cell Signaling Technology; RRID:AB_2145100), BRCA1 (D-9:SCBT; RRID:AB_626761), RAD51 (SCBT:3C10/sc-53428; RRID:AB_630180), γH2AX (Millipore:JBW301/05–636; RRID:AB_309864), GAPDH (Sigma: HPA040067; RRID:AB_10965903), vinculin (Abcam:ab219649; RRID: AB_2819348), β-actin (SCBT:C4; RRID: AB_2714189), and GFP (D5.1:Cell Signaling Technology; RRID:AB_1196615).
3
Western Blot Analysis of TGF-β1 Signaling
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GM tissue (15–20 mg) was homogenized using radioimmunoprecipitation (RIPA) lysis buffer, supernatant was collected after centrifugation, and protein concentration was estimated using a Bio-Rad protein assay (Hercules, CA, USA). Protein samples (25 µg) were loaded and run on Bolt gels (10–15%, Thermo Fisher) for 22 min at 200 V. The gels were transblotted onto iBlot 2 transfer kits using an iBlot 2 Western blot transfer system (Invitrogen, Carlsbad, CA, USA) for 7 min. Membranes were blocked with either 5% non-fat milk or bovine serum albumin (BSA) in 1X TBST (tris-buffered saline, 0.1% Tween-20) at room temperature (RT) for 1h, then incubated with primary antibodies (Table S1A ) for TGF-β1, phosphorylated SMAD2 (pSMAD2), pSMAD3, pSMAD1/5/9, and GAPDH overnight at 4 °C. Following primary antibody incubation, membranes were washed with 1X TBST and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody for 1 h at RT. Finally, membranes were exposed to enhanced chemiluminescence (ECL, Thermo Fisher) reagent and the signal was detected using X-ray films. Densitometric analysis was performed using the ImageJ software on scanned X-ray films. All protein band intensities were normalized to GAPDH and expressed as arbitrary units (a.u.).
4
Western Blot: Standard Protein Analysis
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Following total protein extraction, protein samples were heated to 95 °C for 5 min in the presence of LDS supplemented with 50 mM DTT. 30 μg of total protein was loaded on 4–12% BOLT gels (Thermo Fisher Scientific), separated (200 V, 24 min), and transferred onto polyvinylidene difluoride (PVDF) membranes (activated with methanol; Thermo Fisher Scientific) with transfer buffer (336 mM tris, 260 mM glycine, 140 mM tricine, 2.5 mM EDTA) using a semi-dry Pierce Power Station transfer system (Thermo Fisher Scientific; 25 V, 2.5 mAmp, 10 min). Membranes were blocked for 1 h in milk or BSA (5% w/v in TBS-T: 20 mM TRIS, 1.5 M NaCl, 0.1% (v/v) Tween-20 (pH 7.6)) and incubated in 5% milk or BSA (for phospho- antibodies) containing the appropriate primary antibody (see figure legends; 4 °C, overnight). Membranes were washed extensively with TBS-T and probed with the appropriate secondary antibody in 2.5% milk or BSA (RT, 1 h). Membranes were washed with TBS-T, incubated with ECL substrate (GE Healthcare), and developed using an Odyssey Fc Imaging System (LI-COR). Analysis and quantification were carried out using Image Studio Lite software. For transparency, full, uncropped Western blot images are shown in the Source Data file.
5
Immunoblotting of Phosphorylated Proteins
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Before gel electrophoresis the cell lysate was supplemented with Bolt reducing agent and Bolt gel loading buffer (both Thermo Fisher Scientific, MA, USA) and incubated at 90 °C for 5 minutes. The samples were then separated by SDS-PAGE (Bolt Gels, Thermo Fisher Scientific, MA, USA), followed by transfer onto nitrocellulose membrane, which were blocked in 3% skimmed milk in PBS + 0.1% Tween (PBS-T) for 1 hour at room temperature. Immunoblotting was performed at 4 °C overnight using the following primary antibodies diluted in blocking buffer: phospho/total p44/42 MAPK, phospho/total Akt, phospho/total PLCgamma1 (all at 1:1000 and all from Cell Signaling Technology, USA), or anti-Myc-tag (1:1000; Merck Millipore, MA, USA). The following day the membrane was washed three times in PBS-T and then incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG at 1:5000, from Agilent Technologies, CA, USA) diluted in blocking buffer for 4 hours at 4 °C. This step was followed by another 3 washes in PBS-T and finally chemiluminescent development using the ECL Western Blotting Substrate (Promega, WS, USA). The signal was captured with the BioRad ChemiDoc Imager (Bio-Rad Laboratories, CA, USA) and bands quantified using ImageJ (https://imagej.nih.gov/ij/ ).
6
Immunocapture and Immunoblotting of Tagged Proteins
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Immunoprecipitation was performed using Dynabeads Protein G (Thermo Fisher Scientific) conjugated with antibodies or normal immunoglobulins. GFP-Trap (ChromoTek GmbH, Germany) and anti-HA magnetic beads (Thermo Fisher Scientific) were used to immunocapture EGFP-tagged and HA-tagged proteins, respectively. All protein samples were eluted in reducing Bolt™ LDS Sample Buffer; separated on 4–12% Bolt™ gels (Thermo Fisher Scientific), and electro-transferred to PVDF membranes for immunoblotting of targeting proteins. The signal density was quantified using Image Lab (Bio-Rad, Hercules, CA).
7
Whole Cell Proteome Analysis
8
Western Blot Analysis of Cellular Proteins
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Cellular protein extracts were isolated by directly adding NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 20 mM Tris–HCl [pH 7.4], 5 mM EDTA) containing protease inhibitor cocktail set I (Calbiochem) to the cells after washing twice with ice-cold PBS. Then, the lysates containing cell debris were sonicated by Bioruptor UCD-250HSA (CosmoBio). The soluble fraction was isolated by centrifugation, and protein concentrations of the lysates were measured using DC™ Protein Assay kit (Bio-Rad). Protein extracts were separated by electrophoresis using Bolt™ gels (all blots for immunoprecipitation, and for PIAS3 (protein inhibitor of activated STAT3) and HIF-1α blots) (Thermo) or NuPage™ gels (CAIX, and PRKD2 blots) (Thermo), and then transferred to Immobilon-FL PVDF membranes (Millipore) using Mini Trans-Blot Cell (Bio-Rad). Membranes were blocked for 1 h in BlockPROTM Protein-Free Blocking Buffer (Visual Protein) and probed overnight with primary antibodies for CAIX, AMAP1/ASAP1, PRKD2, PIAS3, HIF-1α or β-actin. Membranes were washed 3 times with TBST and incubated with Alexa fluor® 680- conjugated anti-mouse IgG and/or DyLight® 800 4X PEG-conjugated anti-rabbit IgG for 1 h. Membranes were analyzed using Odyssey® Infrared Imaging System (LI-COR Bioscience).
9
Western Blot Analysis of PrP Protein
10
Western Blot Analysis of Apoptosis Regulators
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Cells were harvested with accutase treatment when adequate, washed with PBS, lysed and heated at 95 °C for 5 min (Laemmli buffer), 70 °C for 10 min (Bolt sample buffer) or 85 °C for 2 min (Tricine sample buffer). Extracts were separated by SDS-PAGE either on 12.5% gels (EZ-run, Fisher Scientific, Schwerte, Germany), 4–12% Bolt gels (Life Technologies) or 16% Tricine gels (Life Technologies), and proteins were transferred onto nitrocellulose membranes (0.2 μm). Membranes were probed with anti-mouse A1 (rat monoclonal, kindly provided by Marco Herold, WEHI, Melbourne, Australia), anti-human A1 (rabbit polyclonal, kindly provided by Jannie Borst), anti-mouse Mcl-1 (Rockland, Limerick, PA, USA, #600-401-394), anti-human Mcl-1 (BD, Biosciences, Heidelberg, Germany, #559027), anti-mouse Bcl-2 (BD, clone 3F11), anti-Bcl-xL (NEB, Frankfurt, Germany, clone 54H6) or anti-GAPDH (Millipore, Darmstadt, Germany, clone 6C5) or anti-β-actin (Sigma, clone AC-15) antibodies. Proteins were visualized using peroxidase-conjugated anti-rabbit (Sigma), anti-mouse (Dianova), anti-hamster (Dianova) or anti-rat (NEB) antibodies by enhanced chemoluminescence detection (ECL Prime, GE Healthcare, Dornstadt, Germany; SuperSignal West Femto Substrate, Pierce, Fisher Scientific, Schwerte, Germany).
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