The whole hearts were cut into small pieces, then lysed on ice with cell lysis buffer supplemented with protease inhibitor and PMSF for 30 min. Then, samples were centrifuged at 12,000 g at 4 °C for 15 min. The concentration of total protein was determined using BCA protein detection kit (Thermo Scientific, Waltham, MA, USA); 50 μg total protein was separated on 10% SDS-PAGE gel, and then transferred to 0.45 μm PVDF membrane (Amersham Hybond, GE Healthcare, München, Germany). The PVDF membrane was blocked with TBST (Tris buffered saline, 0.2% Tween) buffer and 5% milk at room temperature for 2 h, and then incubated at 4 °C overnight with primary antibodies: rabbit anti-TGF-β1 (1:1,000), rabbit anti-Smad2/3 (1:1,000), rabbit anti-p53 (1:1,000), rabbit anti-Bax (1:1,000), rabbit anti-Bcl-2 (1:1,000), rabbit anti-β-actin (1:5,000). After been washed with TBST buffer, the membrane was incubated with goat anti-rabbit secondary antibody at room temperature for 1 h. Subsequently, the membrane was washed with TBST buffer and tested with ECL Kit (ThermoFisher, USA). Protein expression of β-actin was used as an internal control. Image J software (National Institutes of Health, Bethesda, Maryland, USA) was used for gray-scale quantitative analysis.
Amersham hybond
Manufactured by GE Healthcare
Sourced in United States, Germany, United Kingdom
Amersham Hybond is a range of membrane-based products used in molecular biology and genomics applications. These membranes are designed for techniques such as Northern, Southern, and Western blotting, where they serve as a medium for the transfer and immobilization of nucleic acids or proteins. The Amersham Hybond product line offers different membrane types, including nitrocellulose and nylon, to accommodate various experimental requirements.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (18 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Avantor (8 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (5 mentions)
Image lab 6, by Bio-Rad (5 mentions)
Western ip cell lysis buffer, by Beyotime (4 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (4 mentions)
Albumin from bovine serum, by Avantor (3 mentions)
Ab290, by Abcam (3 mentions)
Labtek 2 chambered coverslips, by Thermo Fisher Scientific (3 mentions)
Nupage bis tris gradient gel, by Thermo Fisher Scientific (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Clarity western ecl substrate, by Bio-Rad (3 mentions)
Chemidoc imaging system, by Bio-Rad (3 mentions)
β actin, by Abcam (3 mentions)
Ecl kit, by Thermo Fisher Scientific (3 mentions)
Ab7090, by Abcam (2 mentions)
Ab105767, by Merck Group (2 mentions)
Ab137027, by Abcam (2 mentions)
Protran, by GE Healthcare (2 mentions)
Mab1501, by Abcam (2 mentions)
52 protocols using amersham hybond
1
Western Blot Analysis of Cardiac Proteins
2
Protein Extraction and Western Blot Analysis
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We utilized cell lysis buffer (Beyotime), containing 1% PMSF (Amresco), to cleave proteins for 30 minutes on ice. Then, we centrifuged the lysed cells at 12 000 g for 10 minutes at 4°C to extract the supernatant for protein quantification. We utilized the BCA Protein Assay Kit (Thermo Fisher Scientific, Inc.) to quantify protein concentration. The obtained supernatant was then boiled for 10 minutes by adding 5X SDS. Protein (50 μg) was added to the prepared 12% SDS‐PAGE gels for electrophoretic separation and transferred to 0.45 µm PVDF membranes (Amersham Hybond, GE Healthcare). We then utilized 1% albumin from bovine serum (Amresco) to block the PVDF membranes for 2 hours. Then, the membranes were incubated overnight with diluted xCT (1:1000, ab37185; Abcam) and β‐actin (1:1000, ab179467; Abcam) antibodies on a shaker at 4°C. We washed the membranes with TBS‐T (0.1% Tween‐20) at room temperature three times for 10 minutes. The goat anti‐rabbit IgG H&L (HRP) (1:2000, ab7090; Abcam) for 1 hour was utilized to incubate the membranes. After washed, the membranes were exposed to enhanced chemiluminescence substrate detection solution (Lulong Biotech) subsequently.
3
Total Yeast RNA Preparation and Analysis
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Total yeast RNA preparation was carried out using the procedure described by Lim et al. (2003 ). RNA concentrations were determined using 96 microplates in the Multiskan GO UV spectrophotometer (Thermo Fisher Scientific Inc.) and approximately 30 µg of total RNA from each yeast transformant was analyzed on 1.2% (w/v) denatured formaldehyde-agarose gel. RNAs were blotted onto an Amersham Hybond™ nylon membrane (GE Healthcare) according to the manufacturer’s instructions. Hybridization was carried out in modified Church buffer (Nguyen et al. 2013 (link)) with a scEDIII-PIGS PCR product as a detecting probe.
4
Western Blot Analysis of GFP Expression
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Expression of GFP was analyzed in automiG induced cells by western blotting using mouse anti-GFP (Roche®) and anti-Mbf1 antibodies (66 (link)) as loading and transfer control. Seventy two hours after the dsRNA transfection and automiG vector induction, the culture medium was removed and 80 μl of Sample Buffer Laemmli 2X (Sigma®) was added in each well. The samples were boiled (95°C) and 18 μl were loaded onto a 4–20% Mini-PROTEAN®TGX™ 12 well-gel (Bio-Rad). After transfer onto a PVDF (Amersham Hybond, GE Healthcare) or nitrocellulose membrane, membranes were blocked in 5% milk, dissolved in 1× TBS-T (20 mM Tris-Base, 150 mM NaCl, Tween-20 (Polyoxyethylene sorbitane monolaureate) to 0.05%) and incubated overnight with anti-GFP (1:2000) or anti-Mbf1 (1:10.000) antibodies diluted in the blocking solution. After three times 15 min washes, appropriate secondary antibody (1:10 000) coupled to alkaline phosphatase (Promega) was added and incubated for one hour at room temperature. Detection was performed using BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and NBT (nitro-blue-tetrazolium, (ThermoFischer) reagents diluted in AP buffer (100 mM Tris–HCl pH 9.5, 100 mM NaCl, 5 mM MgCl2).
5
Western Blotting of ESCRT Proteins
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Cells were seeded and transfected as described above in LabTek II chambered coverslips (ThermoScientific). Protein extracts were separated on 4-12% NuPAGE Bis-Tris gradient gels (Life Technologies) and transferred to Nitrocellulose (Protran, GE Healthcare) or PVDF membranes (Amersham Hybond, GE Healthcare). Western blotting was performed by standard methods using antibodies against CHMP4B (1:1000, Abcam, ab105767), actin (1:30000, Merck Millipore, MAB1501), VPS4B (1:500, Abcam, ab137027), GFP (1:5000, Abcam, ab290) and GAPDH (1:2500, Abcam, ab9485).
6
Western Blot Analysis of Protein Expression
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Tissues or cells were lysed in Western & IP cell lysis buffer (Beyotime) with PMSF (Amresco) for 30 minutes on ice at 4°C followed by centrifugation at 12 000 × g for 15 minutes at 4°C. The supernatants were collected, and the total protein concentration was measured using the BCA Protein Assay Kit (Thermo Scientific). Equimolar amounts of protein were loaded into each well and separated with 12% SDS‐PAGE. Then, proteins were transferred to a 0.45‐μm PVDF membrane (Amersham Hybond, GE Healthcare), which was blocked in 2% bovine serum albumin (Amresco) prior to overnight incubation overnight at 4°C with the following primary antibodies: rabbit anti‐IL‐1RA, rabbit anti‐IL‐1α (1:1000), mouse anti‐β‐actin (1:2000; Cell Signaling Technology), and VEGF‐A polyclonal antibody (1:1000, A41552). After three washes in TBST buffer lasting 10 minutes per wash, the membrane was incubated with secondary antibodies for 1 hour at room temperature. The blots were developed using enhanced chemiluminescence (Lulong Biotech).
7
Immunoprecipitation and Western Blotting Protocol
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Cell lysates were incubated with specific antibodies and lysis buffer for 4 hr. Subsequently, 30 μl of washed Dynabeads (14311D, Thermo Fisher, Waltham, MA) were added to each lysate and incubated overnight at 4°C. Next, the beads were washed five times, and the antigens were eluted twice using 8 M urea buffer (8 M urea, 20 mM Tris pH 7.5, and 100 mM NaCl) and concentrated. The resulting samples were separated by Mini-PROTEAN TGX (4–20%, 4561093, Bio-Rad Laboratories, Hercules, CA) and transferred onto nitrocellulose membranes (Amersham Hybond, 10600021, GE Healthcare) using Trans-Blot SD Semi-dry Transfer Cell system (Bio-Rad Laboratories). The membranes were then blocked with 5% skim milk in Tris-buffered saline + Tween-20 (TBS-T; 20 mM Tris, 137 mM NaCl, 0.1% Tween-20, pH 7.6), incubated overnight at 4°C with a 1:1000 dilution of each antibody, and subsequently incubated for 1 hr with a 1:5000 dilution of a horseradish peroxidase–conjugated goat anti-mouse secondary antibody (ab6789, Abcam, Cambridge, UK) or goat anti-rabbit secondary antibody (ab6721, Abcam). Immunoreactive proteins were detected using SuperSignal West Dura Extended Duration Substrate (34076, Thermo, Rockford, IL) and detected using a ChemiDoc MP Imaging system (Bio-Rad Laboratories). The band intensity was densitometrically evaluated using Image Lab software (version 5.2, Bio-Rad Laboratories).
8
ALCAR Induces Apoptosis in Prostate Cells
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ALCAR ability to induce apoptosis in PCa and BPH cells was confirmed by western blotting. Following 24 h of treatment with ALCAR (1 or 10 mM), cells were lysed in RIPA buffer, supplemented with protease and phosphatase inhibitor cocktails (Roche Diagnostics GmbH). Proteins (30 μg) were separated on the NupageNovex on 4–12% Bis-Tris Gel (Life Technologies) and transferred to a PVDF membrane Amersham Hybond (GE Healthcare Biosciences). Membranes were incubated overnight at 4 °C with Cleaved Caspase-3 (Asp175) (Cell Signalling Technology) and with peroxidase-linked anti-rabbit IgG or anti-mouse IgG secondary antibodies (GE Healthcare Life science) for 1 h at room temperature. Specific protein bands were detected with Pierce ECL Western Blotting Substrate (ThermoFisher Scientific). Protein expressions were normalized to beta-Actin (Abcam). Band intensity (revealed as optical density-OD) were detected by ImageJ software.
9
Western Blot Protein Analysis
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For western blots 2 × 106 cells were resuspended in 200 μl 2x Laemmli Sample Buffer (0.125 M Tris HCl, 20% glycerol, 4% SDS and 0.002% Bromphenol Blue). Lysates were homogenized through a 23 gauge needle and denatured at 70 °C for 10 min. Between 18 and 25 μl of protein lysate was loaded on 12% SDS-PAGE and transferred onto a PVDF membrane (GE Healthcare Amersham™ Hybond™). After incubation with the specific primary antibody (see Supplementary Information ), the membranes were incubated with appropriate secondary peroxidase-conjugated antibodies. For detection ECL™ Prime Western Blotting System (GE Healthcare, Chicago, IL, USA) and the Gel Logic 1500 imaging system analyzed with Kodak Molecular Imaging Software (Version 5.0) was used.
10
STAT3 Phosphorylation Evaluation in Cancer
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We assessed the A009 extract ability to target STAT3 phosphorylation, as a biochemical consequence of their cancer preventive and angiopreventive properties. Following 24 h of treatment with A009 extract (1:250, batch 4 or 3) or HyT alone at the same concentration present in A009 dilutions, the cells were lysed in RIPA buffer, supplemented with protease and phosphatase inhibitor cocktails (Roche Diagnostics GmbH). Proteins (50 μg) were separated on the NupageNovex on 4–12% Bis-Tris Gel (Life Technologies) and transferred to a PVDF membrane Amersham Hybond (GE Healthcare Biosciences). Membranes were incubated overnight at 4 °C with anti-p-STAT3 (Tyr705) (Cell Signaling Technology) and with peroxidase-linked anti-rabbit IgG secondary antibodies (GE Healthcare Life science) for 1 h at room temperature. Specific protein bands were detected with Pierce ECL Western Blotting Substrate (ThermoFisher Scientific). Band intensity (revealed as optical density-OD) was quantified using ImageJ software, v 1.52. Every band was normalized versus the respective housekeeping (HK) protein (beta-actin for A549 cells and tubulin for H1650 cells). Finally, HK normalized bands, were further normalized versus not-treated (NT) cells.
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