Specific proteins were analyzed with epitope-specific primary antibodies to phospho-JNK, JNK, phospho-ERK, ERK, phospho-p38, p38, phospho-IKKα/β, IKKα (IκB kinase alpha), IKKβ (IκB kinase beta), phospho-I-κBα, I-κBα (inhibitor of kappa B alpha), phospho-NF-κB p65, NF-κB p65, iNOS, COX-2, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and horseradish peroxidase (HRP)-conjugated anti-rabbit antibodies (Cell Signaling, Danvers, MA, USA) using western blotting. Equal amounts of protein samples (20 μg total protein per lane) were separated by electrophoresis on a 10% sodium dodecyl sulfate-polyacrylamide gel and then transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was incubated with epitope-specific primary antibodies overnight at 4 °C, followed by incubation with HRP-conjugated anti-rabbit antibodies for 1 h at room temperature. The bound antibodies were developed using enhanced chemiluminescence (ECL) Advance Western Blotting Detection Reagents (GE Healthcare, Little Chalfont, UK) and scanned with a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Germany).
Ecl advance western blotting detection reagent
Manufactured by GE Healthcare
Sourced in United Kingdom
ECL Advance Western Blotting Detection Reagents are a chemiluminescent substrate solution designed for the detection of proteins on western blots. The reagents generate a luminescent signal in the presence of the horseradish peroxidase (HRP) enzyme, which is commonly used to label antibodies in western blot procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Cell Signaling Technology (10 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Mini protean tgx gel, by Bio-Rad (7 mentions)
Las 4000 imaging system, by Fujifilm (5 mentions)
Fusion solo chemiluminescence system, by Avantor (5 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Las 4000 film, by Fujifilm (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Hoechst 33342, by Merck Group (2 mentions)
Leupeptin, by Cell Signaling Technology (2 mentions)
Na3vo4, by Cell Signaling Technology (2 mentions)
Charcoal dextran stripped human serum, by Innovative Research (2 mentions)
Rpmi 1640 medium, by Corning (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Keap1, by Abcam (2 mentions)
Hrp conjugated anti rabbit secondary antibody, by Santa Cruz Biotechnology (2 mentions)
β actin, by Abcam (2 mentions)
Iodixanol, by Merck Group (2 mentions)
Phospho jnk, by Cell Signaling Technology (2 mentions)
32 protocols using ecl advance western blotting detection reagent
1
Protein Signaling Pathway Analysis
2
Western Blot Analysis of Whole Cell Proteins
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Proteins (whole cell extracts, 30 μg/lane) were separated by electrophoresis in a precast 4–15% Mini-PROTEAN TGX gel (Bio-Rad, CA, USA) blotted onto PVDF transfer membranes as reported previously [26 (link)]. Bound antibodies were visualized using ECL Advance Western Blotting Detection Reagents (GE Healthcare, UK) and a LAS 4000 imaging system (Fujifilm, Japan).
3
Western Blot Analysis of Protein Signaling
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The same amount of protein from each of the total proteins, cytoplasmic extractions, and nuclear fractions was transferred to a polyvinylidene difluoride transfer membrane from a precast 4–15% Mini-PROTEAN TGX gel (Bio-Rad, Hercules, CA, USA). The membranes were exposed to primary antibodies, including Nrf2, heme oxygenase 1 (HO-1), phospho-IκB kinase α/β (p-IKKα/β), IκB kinase α (IKKα), IκB kinase β (IKKβ), phospho-inhibitor of kappa B alpha (p-IκBα), inhibitor of kappa B alpha (IκBα), nuclear factor kappa B (NF-κB), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), lamin B, and GAPDH, followed by horseradish peroxidase-conjugated secondary antibodies. All the antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The detection was performed using ECL Advance Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK) and a Fusion Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany). The signal intensity of each band was quantified using Fusion-Capt v16.10 software (PEQLAB Biotechnologie GmbH).
4
Western Blot Analysis of NF-κB Pathway
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Proteins in the samples (20 μg protein/lane) were separated by electrophoresis on a 10% sodium dodecyl sulfate–polyacrylamide gel and further transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was incubated with primary antibodies (Cell Signaling, Beverly, MA, USA) against IKKα, phospho-IKKα/β, IKKβ, I-κBα, phospho-I-κBα, NF-κB p65, phospho-NF-κB p65, iNOS, COX-2, and GAPDH for 1 h at room temperature. After binding with HRP-conjugated anti-rabbit antibodies (Cell Signaling, Beverly, MA, USA) for 1 h, the PVDF membranes were developed using enhanced chemiluminescence (ECL) Advance Western Blotting Detection Reagents (GE Healthcare, Little Chalfont, UK) and visualized using a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Germany).
5
Western Blot Analysis of Cellular Proteins
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Cells (8 × 105 cells) grown in 60 mm dishes were treated with the indicated concentration of samples for 24 h. Whole-cell extracts were then prepared according to the manufacturer's instructions using RIPA buffer (Cell Signaling, MA, USA) supplemented with 1x protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF) [23 (link)]. Proteins (whole-cell extracts, 30 μg/lane) were separated by electrophoresis in a precast 4–15% Mini-PROTEAN TGX gel (Bio-Rad, CA, USA), blotted onto PVDF transfer membranes, and analyzed with epitope-specific primary and secondary antibodies. Bound antibodies were visualized using ECL Advance Western Blotting Detection Reagents (GE Healthcare, UK) and a LAS 4000 imaging system (Fujifilm, Japan).
6
Western Blot Analysis of Autophagy Proteins
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Whole-cell lysates were prepared by incubating cells in RIPA buffer (Beverly, MA, USA) supplemented with protease inhibitor cocktail (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, cells were harvested and collected by centrifugation at 13,200 rpm for 5 min and washed in Dulbecco’s Phosphate-Buffered Saline (DPBS) (pH 7.2). The pellets were solubilized in the same volume of mitochondrial lysis buffer for 5 min and then centrifuged at 13,200 rpm for 20 min at 4°C. Equal amounts of lysate protein were loaded and separated on a 15% SDS-PAGE gel. Proteins were electrophoretically transferred to a PVDF membrane, and the membrane was blocked in 5% skim milk in tris-buffered saline containing 0.1% Tween-20 for 1 h. Then, the membranes were incubated in the presence of following primary antibodies (LC3, beclin-1, mTOR, AMPK, GAPDH from cell signaling (Beverly, MA, USA); GATE16, TP53INP2/DOR from Epitomics (Burlingame, CA, USA)) at 4°C overnight. The membranes were then washed three times with TBS Tween 20 and probed with the corresponding secondary antibodies conjugated with HRP at room temperature for 1 h. Detection was carried out using an enhanced ECL Advance Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK) followed by LAS-4000 film (Fujifilm, Tokyo, Japan).
7
Western Blotting of Target Proteins
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Western blotting analysis was used to assess expression of target proteins. Briefly, after treatment, the harvested cells were washed twice with cold phosphate-buffered saline, and total cell lysates were prepared with radio immunoprecipitation assay buffer (RIPA buffer, Cell Signaling Technology, Inc., Beverly, MA, USA) supplemented with 1 × EDTA-free protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF) according to the manufacturer’s instructions.
Protein content was quantified via bicinchoninic acid (BCA) protein assay, and 20 μg, along with molecular weight markers, was separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), for 90 min at 110 V. This was then transferred to a polyvinylidene fluoride (PVDF) transfer membrane and immunoblotted with corresponding antibodies. Immunodetection was performed using the ECL Advance Western Blotting Detection Reagents (GE Healthcare, Cambridge, UK) and a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany) [59 (link)].
Protein content was quantified via bicinchoninic acid (BCA) protein assay, and 20 μg, along with molecular weight markers, was separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), for 90 min at 110 V. This was then transferred to a polyvinylidene fluoride (PVDF) transfer membrane and immunoblotted with corresponding antibodies. Immunodetection was performed using the ECL Advance Western Blotting Detection Reagents (GE Healthcare, Cambridge, UK) and a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany) [59 (link)].
8
Hypoxylonol F Protects Renal Cells from Cisplatin Toxicity
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The protective effect of hypoxylonol F (2 ) against cisplatin-induced renal cellular pathway was determined by Western blotting analysis [28 (link)]. LLC-PK1 cells were seeded into 6-well culture plates (4 × 105 cells/well) and treated with hypoxylonol F (2 ) as described above, washed twice with cold phosphate-buffered saline. Cells were scraped from the plates and lysed in RIPA buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) supplemented with 1× EDTA-free protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF) according to the manufacturer’s instructions. A bicinchoninic acid protein assay was used for the quantitation of protein of samples, and 20 μg of protein were separated using 10% SDS PAGE gels for 90 min at 80 V. The proteins were transferred to PVDF transfer membranes, which were incubated with primary antibodies against phospho-p38, p38, phospho-JNK, JNK, phospho-ERK, ERK, cleaved caspase-3, and GAPDH (1:1000 dilution). Horseradish peroxidase-conjugated anti-rabbit IgG (1:2000 dilution) was used to detect the primary antibodies. Bound antibodies were visualized using ECL Advance Western Blotting Detection Reagents (GE Healthcare, Cambridge, UK). GAPDH was utilized as a loading control.
9
Investigating Cisplatin-Induced Apoptosis
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Cells were seeded at a density of 4 × 105 cells per/well in 6-well plates. One day after seeding, cells were treated for 2 h with the test samples. Then, 25 μM cisplatin was added to wells for 24 h. Untreated cells were used as controls. After incubation, cells were washed twice with cold phosphate-buffered saline and scraped off the plates, and then lysed using RIPA buffer (Cell Signaling Technology, Inc., Beverly, MA, USA) supplemented with 1× EDTA free protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF) according to the manufacturer’s instructions. Aliquots of 20 μg protein were subjected to SDS PAGE (10% gels) along with molecular weight markers for 90 min at 110 V, and then transferred to PVDF transfer membranes. Membranes were probed with primary antibodies to phospho-JNK, JNK, phospho-p38, p38, cleaved caspase-3, GAPDH and then with secondary immunoglobulin G horseradish peroxidase conjugates. Bound antibodies were detected using ECL Advance Western Blotting Detection reagents (GE Healthcare, Cambridge, UK) and a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany).
10
FBG Extract Effects on HUVECs
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HUVECs (8 × 105 cells/mL) grown in 60 mm dishes were treated with the various concentrations of FBG extract (12.5 μg/mL and 25 μg/mL) for 24 h. Next, cell extracts were prepared using RIPA buffer (Cell Signaling, Danvers, MA, USA) that was supplemented with 1× protease inhibitor cocktail and 1mM phenyl methyl sulfonyl fluoride. Proteins (30 μg/lane) were separated by electrophoresis, transferred onto polyvinylidene fluoride membranes, and allowed to bind with epitope-specific primary and secondary antibodies. Visualization of antibody bounding was confirmed using ECL Advance Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK) and a LAS 4000 imaging system (Fujifilm, Tokyo, Japan) following instruction manual.
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