Cell lysates were prepared in 2X Laemmli sample buffer (Bio-Rad) containing protease and phosphatase inhibitors (Roche) and denatured at 95°C for 5 min. Samples were run on 10 or 15% SDS-PAGE gels and transferred onto PVDF membranes (GE Healthcare) using standard techniques. Immunoblots were blocked in PBS containing 5% non-fat milk and 0.1% Tween 20 at room temperature for 1 h and then incubated with primary antibodies overnight at 4°C. After extensive washes, immunoblots were incubated with horseradish peroxidase-conjugated secondary antibodies (Dako), and proteins were visualized by enhanced chemiluminescence (Radiance Plus, Azure) acquired on Azure Biosystems Imaging System (Cambridge Bioscience).
Radiance plus
Manufactured by Azure Biosystems
Sourced in United States
Radiance Plus is a multipurpose spectrofluorometer designed for a variety of fluorescence-based applications. It features high-performance optics and a modular design to accommodate different experimental needs.
Automatically generated - may contain errors
Lab products found in correlation
Azure c600, by Azure Biosystems (3 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Radiance peroxide, by Azure Biosystems (2 mentions)
Fusion fx, by Vilber (2 mentions)
Immobilon p pdvf membrane, by Merck Group (2 mentions)
Immobilon western hrp substrate, by Merck Group (2 mentions)
A0545, by Merck Group (2 mentions)
Gtx113340 10, by GeneTex (2 mentions)
Anti phospho akt, by Cell Signaling Technology (2 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (2 mentions)
Anti phospho p70s6k, by Cell Signaling Technology (2 mentions)
Anti phospho ampkα, by Cell Signaling Technology (2 mentions)
Dc protein assay kit, by Bio-Rad (2 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Tbs t, by Santa Cruz Biotechnology (1 mentions)
Fast semi dry blotter, by Thermo Fisher Scientific (1 mentions)
13 protocols using radiance plus
1
Western Blot Analysis of Protein Samples
2
Western Blot Analysis of Aryl Hydrocarbon Receptor
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Total protein was isolated in parallel with large RNA and small RNA with the NucleoSpin® miRNA kit (Macherey-Nagel) according to the manufacturer’s protocol. Protein pellets were resuspended in protein solving buffer set PSB/TCEP (Macherey-Nagel). Proteins were loaded and separated in a 10% acrylamide gel (100v) and transferred onto a 0.2-μm nitrocellulose membrane (Amersham Protran, Buckinghamshire, UK) using the Thermo-Pierce Fast Semi-Dry Blotter apparatus. The membrane was blocked with 5% nonfat milk for 1 h at room temperature with agitation, followed by overnight incubation at 4°C with primary antibodies against aryl hydrocarbon receptor (AHR) (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and GAPDH (1:1,000; Santa Cruz Biotechnology) in BSA with tris–buffered saline–Tween 2 (TBS-T; Santa Cruz Biotechnology). Subsequently, the membrane was washed and incubated with secondary antibody (1:2,000; Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature. Protein signal was detected with enhanced chemiluminescence horseradish peroxidase (HRP) substrate (Radiance Plus; Azure Biosystems, Dublin, CA, USA) and recorded using the Azure Biosystems c600 quantitative Western blot imaging system (Azure Biosystems).
3
Versatile Western Blot Visualization
4
Immunoblot Analysis of Fibroblast Proteins
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Immunoblot analysis was carried out as previously described [71 ]. Briefly, total protein was extracted from fibroblasts using RIPA buffer with Pierce Protease Inhibitor and protein concentration was measured using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). 7.5 µg of protein per sample, and a 4–20% Tris–glycine gradient gel were used for SDS-PAGE (Bio-Rad, Hercules, CA, USA). Protein was transferred to a polyvinylidene fluoride membrane. 5% bovine serum albumin in PBST was used for all blocking steps (Equitech-Bio, Kerrville, TX, USA). All primary antibodies were incubated in blocking solution overnight at 4 °C. Information about antibodies is detailed in Additional file 1 . Secondary antibodies were incubated for 1 h at room temperature. Restore Western Blot Stripping Buffer was used to strip the membranes (Thermo Fisher Scientific). Membranes were visualized by Radiance Peroxide and Radiance Plus (Azure Biosystems, Dublin, CA, USA). Immunoreactive proteins were imaged using the Azure c600 (Azure Biosystems).
5
Immunoblotting Analysis of Arf Proteins
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Cell lysates were denatured in SDS-sample buffer for 5 min at 95°C (10 min at 37°C to analyze KDEL receptors), separated by SDS-gel electrophoresis (15% polyacrylamide for Arfs), and transferred to Immobilon-P PDVF membranes (Millipore). Membranes were blocked with TBST (20 mM Tris, 150 mM NaCl, pH 7.6, and 0.1% Tween20) with 3% nonfat dry milk for 1 h and incubated with primary antibody in TBST with 1% milk overnight at 4°C: anti-Arf1 (1:2,500; MAB10011; Abnova), anti-Arf3 (1:1,000; 610784; BD Bioscience), anti-Arf4 (1:1,000; 11673–1-AP; Proteintech), anti-Arf5 (1:750; H00000381-M01; Abnova), anti-actin (1:100,000; MAB1501; Sigma-Aldrich), anti-calreticulin (1:2,500; 27298–1-AP; Proteintech), anti-Grp78/BiP (1:10,000; GTX113340-10; Genetex), and anti-KDEL receptor antibody (1:1,000; 69659; Abcam). After washing, the membranes were incubated with HRP-conjugated secondary antibody (1:10,000, anti-rabbit, A0545, Sigma-Aldrich; anti-mouse, A0168, Sigma-Aldrich) in TBST with 1% milk. Chemiluminescence signals were detected using Immobilon Western HRP Substrate (Millipore) or Radiance Plus (Azure Biosystems) and imaged using a FusionFX (Vilber Lourmat).
6
Angiotensin II Signaling in EC219 Cells
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EC219 cells were grown in RPMI medium containing 10% FCS and antibiotics until 95% confluence. Then 0.1 µM angiotensin (Ang) II (Bachem), 10 µM losartan (Sigma-Aldrich), and 100 µM SS-31, either alone or in combination, were added for 24 h. The cell layers were washed in PBS and the pellets suspended in the SDS-PAGE protein loading buffer. SDS-PAGE electrophoresis was performed in 10% gels, and the proteins were transferred to a PVDF membrane (Bio-Rad Laboratories). The membranes were probed sequentially with a rabbit monoclonal anti-AT1R antibody (clone ab124734, dilution 1/500; Abcam), followed by an anti-rabbit IgG-HRP antibody (dilution 1/1000; Cell Signaling Technology) and Radiance Plus (Azure Biosystems, Dublin, CA). The whole process was repeated on the same membrane with the anti-AT2R antibody (Abcam; ab92445, dilution 1/500) and the anti-β-actin antibody (clone 4967, dilution 1/2500; Cell Signaling Technology). Images of chemiluminescence were taken using FUSION-FX (Fisher Biotec). Membranes were analyzed using Photofiltre software and GelAnalyzer software (lazarsoftware).
7
Western Blot Analysis of STAR Protein
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Protein was extracted from mouse and rat primary Leydig cells, WT MA-10 cells, and STAR KO cells using RIPA buffer. Protein concentration was determined after centrifugation using the Pierce BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using 7.5 μg of protein extract and a 4%–20% Tris–glycine gradient gel (Bio-Rad, Hercules, CA, USA) and the resulting bands electro-transferred to a polyvinylidene fluoride membrane. Blocking of the membranes was performed using 5% bovine serum albumin before incubating with STAR antibody (1:1000; Cell Signaling, Danvers, MA, USA) and secondary antibody, WesternSure® Goat anti-Rabbit HRP Secondary Antibody (1:5000; LI-COR, Lincoln, NE, USA). Membranes were stripped using the Restore Western Blot Stripping Buffer (Thermo Scientific, Waltham, MA, USA) and reprobed using anti-β-actin (1:5000). The immunoreactive proteins were visualized using Radiance Peroxide and Radiance Plus (Azure Biosystems, Dublin, CA, USA) and imaged using the Azure c600 (Azure Biosystems).
8
Western Blot Protocol for Protein Analysis
9
Protein Extraction and Western Blot Analysis from Brain Samples
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Protein was extracted from the cell layer that was collected after metabolite extraction of brain samples, and protein quantification and Western blots were done as previously described [16 (link)]. The following antibodies from Cell Signaling Technology (Danvers, MA, USA) were used for the western blots: rabbit anti-Akt (#9272), anti-phospho-Akt (#9275; Thr308), anti-AMPKα (#2603), anti-phospho-AMPKα (#2535; Thr172), anti-p70 S6K (#9202), anti-phospho-p70 S6K (#9234; Thr389), as well as goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (#7074). Either Clarity Western ECL Blotting Substrates (Bio-Rad) or Radiance Plus (Azure biosystems, Dublin, CA, USA) were used depending on the strength of signal for chemiluminescent detection. Refer to Appendix A for more detailed description.
10
Ni-NTA Affinity Purification of Recombinant Proteins
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Nickel-nitrilotriacetic acid (Ni-NTA) chromatography resin, Thermo Scientific #88221. Protein G agarose, Protein Mods LLC, IgG binding capacity 20 mg/mL #PGG. CNBr-activated Sepharose 4-Fast Flow. Amersham Biosciences #17-0981-01. C18 spin columns Pierce #89870. Steriflip vacuum filtration system 0.22 μm membrane Millipore #SCGP00525. Trypsin sequencing grade modified, Promega #V5113. Precast polyacrylamide gels Bio-Rad #4561044 (12%). Polyvinylidene fluoride (PVDF) 0.45 μm Immobilon-P transfer membrane, Millipore #IPVH00010. Enhanced Chemiluminescence (ECL) reagents Radiance Plus, Azure Biosystems #AC2103. Affinity purified rabbit anti-denatured Usp polyclonal, produced in-house. Donkey anti-rabbit IgG conjugated to horse radish peroxidase (HRP), Jackson ImmunoResearch #711-035-152. Bicinchoninic acid (BCA) protein assay, Pierce #23255. pET28a, Novagen #69864–3. 2x Yeast extract tryptone culture medium (2YT), Sigma #Y2377.
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