Enhanced chemiluminescence detection kit

Manufactured by GE Healthcare

Sourced in United Kingdom, United States, Australia, Sweden, Italy

The Enhanced Chemiluminescence Detection Kit is a laboratory equipment product that enables the detection and quantification of proteins in Western blot analysis. The kit utilizes a chemiluminescent substrate to generate a luminescent signal that can be captured and measured, providing a sensitive and reliable method for protein detection.

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Lab products found in correlation

β actin, by Merck Group (9 mentions) Pvdf membrane, by Merck Group (9 mentions) Nitrocellulose membrane, by Bio-Rad (6 mentions) Polyacrylamide gel, by Bio-Rad (5 mentions) Nitrocellulose membrane, by Bio Basic (4 mentions) Protease inhibitor cocktail, by Merck Group (4 mentions) Pierce coomassie plus colorimetric protein assay, by Thermo Fisher Scientific (4 mentions) Polyvinylidene fluoride membrane, by Bio-Rad (4 mentions) Las 4000, by Cytiva (4 mentions) Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (3 mentions) Quantity one 4, by Bio-Rad (3 mentions) Ripa buffer, by Merck Group (3 mentions) Nitrocellulose membrane, by Thermo Fisher Scientific (3 mentions) Pvdf membrane, by GE Healthcare (3 mentions) Ripa lysis buffer, by Cell Signaling Technology (3 mentions) Horseradish peroxidase conjugated to appropriate secondary antibodies, by Bio-Rad (3 mentions) Polyvinylidene difluoride membrane, by Merck Group (3 mentions) Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (3 mentions) Polyvinylidene fluoride membrane, by Merck Group (3 mentions) Radioimmunoprecipitation assay buffer, by Merck Group (2 mentions)

Close spelling variants of this product

Enhanced chemiluminescence (612 citations) Enhanced chemiluminescence kit (198 citations) Enhanced chemiluminescence detection (42 citations) Chemiluminescence detection kit (31 citations) Chemiluminescence kit (29 citations) ECL enhanced chemiluminescence detection kit (24 citations) Enhanced Chemiluminescence Western Blotting Detection kit (4 citations) Amersham enhanced chemiluminescence Western blotting detection kit (3 citations) Enhanced Chemiluminescence Detection kit reagents (2 citations) Enhanced Chemiluminescence Select Western Blotting detection kit (2 citations)

115 protocols using enhanced chemiluminescence detection kit

Mitochondrial Cytochrome C Release Assay

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Treated cells were lysed using radioimmunoprecipitation assay buffer (Sigma-Aldrich,
USA) containing 1% protease inhibitor cocktail and 2% phenylmethanesulfonyl fluoride.
Detection of Cyto C release from mitochondria to cytoplasm was performed using
cytoplasmic extracts prepared as described previously (23 (link)). The supernatants were collected, and protein concentrations
were determined by the Bradford method. Proteins were separated using 12% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and transferred
electrophoretically onto nitrocellulose membranes (Bio Basic, USA). The transferred
membranes were then blotted with antibodies as follows: phospho-ERKs (P-ERKs),
total-ERKs (T-ERKs), phosphor-AKT (P-AKT), total-AKT (T-AKT), Bcl-2, Bax, cleaved
caspase 3, Cyto C, and glyceraldehyde-3-phosphate dehydrogenase (all 1:1,000; Cell
Signaling, USA) at 4°C overnight, followed by treatment with horseradish
peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, USA). The
signals were detected with enhanced chemiluminescence detection kits (GE Healthcare,
UK). The intensities of the bands were quantified with scanning densitometry using
the Quantity One 4.5.0 software (Bio-Rad).

Wang D., Guo T.Q., Wang Z.Y., Lu J.H., Liu D.P., Meng Q.F., Xie J., Zhang X.L., Liu Y, & Teng L.S. (2014). ERKs and mitochondria-related pathways are essential for glycyrrhizic acid-mediated neuroprotection against glutamate-induced toxicity in differentiated PC12 cells. Brazilian Journal of Medical and Biological Research, 47(9), 773-779.

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Neuroprotective Role of 5zou in 6-OHDA/L-Glu-Induced Cytotoxicity

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DPC12 cells were pre-treated with 20 and 40 µM 5zou for 3 h, and followed with 24-h incubation of 6-OHDA (100 µM) and L-Glu (25 mM). Cells were lysed by radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck KGaA) containing 2% phenylmethanesulfonyl fluoride (Sigma-Aldrich; Merck KGaA) and 1% protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). Protein concentrations were determined using a Standard BCA Protein Assay kit (Merck KGaA). Proteins (30 µg) were separated on a 12% SDS-PAGE gel and electrophoretically transferred onto nitrocellulose membranes (Bio Basic, Inc., Markham, ON, Canada). The membranes were blocked in 5% bovine serum albumin at room temperature for 4 h, and then incubated with the following primary antibodies (all diluted 1:1,000) overnight at 4°C: Bcl-2 (ab321224), Bcl-xL (ab7973), and GAPDH (ab8245) (1:1,000; Abcam, Cambridge, UK) at 4°C overnight, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (sc-2005 and sc-358925) at dilution of 1:2,000 at 4°C for 4 h (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Chemiluminescence was detected by using enhanced chemiluminescence detection kits (GE Healthcare Life Sciences, Little Chalfont, UK).

Wang D., Hu S., Zhang J., Li Q., Liu X, & Li Y. (2017). Investigation of the neuroprotective effects of a novel synthetic compound via the mitochondrial pathway. Molecular Medicine Reports, 16(2), 1133-1138.

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Western Blot Analysis of Signaling Proteins

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Treated cells were lysed in radioimmunoprecipitation assay buffer containing 1% protease inhibitor cocktail and 2% phenylmethanesulfonyl fluoride (Sigma-Aldrich). In order to detect cytochrome c (cyto c) release, cytoplasmic extracts were prepared as described previously by Yang et al (20 (link)). A total of 30 μg protein was separated using 10–12% SDS-PAGE and electrophoretically transferred onto nitrocellulose membranes (pore size, 0.45 μm; Bio Basic, Inc., Markham, ON, Canada). The transferred membranes were then blotted with antibodies against phosphorylated (P)-ERKs, total (T)-ERKs, P-AKT, T-AKT, P-glycogen synthase kinase-3β (GSK3β), T-GSK3β, B-cell lymphoma 2 (Bcl-2), Bcl2-associated X protein (Bax), cyto c and GAPDH at dilutions of 1:1,000 (Cell Signaling Technology, Inc., Danvers, MA, USA) at 4°C overnight. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) for 3 h at 4°C. Chemiluminescence was detected using enhanced chemiluminescence detection kits (GE Healthcare, Amersham, UK). The intensity of the bands was quantified by scanning densitometry using Quantity One 4.5.0 software (Bio-Rad Laboratories, Inc.).

TENG L., MENG Q., LU J., XIE J., WANG Z., LIU Y, & WANG D. (2014). Liquiritin modulates ERK- and AKT/GSK-3β-dependent pathways to protect against glutamate-induced cell damage in differentiated PC12 cells. Molecular Medicine Reports, 10(2), 818-824.

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Western Blot Analysis of LC3 and P-AKT

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Protein concentrations were determined with the Bradford method (Redmile-Gordon et al., 2013). Proteins were separated by 12% SDS-PAGE and transferred electrophoretically onto nitrocellulose membranes (Bio Basic, New York, USA). The membranes were then reacted with rabbit anti-LC3 polyclonal antibody or rabbit anti-P-AKT polyclonal antibody (both 1:1,000; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight, followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1,000; Santa Cruz Biotechnology, Dallas, TX, USA) at 37°C for 12 hours. The signals were detected with enhanced chemiluminescence detection kits (GE Healthcare, Chicago, IL, USA). The gray scales of the bands were quantified by scanning densitometry using Quantity One 4.5.0 software (Bio-Rad, Hercules, CA, USA).

Liu Y., Liu L., Ying X.X., Wei W.J., Han C., Liu Y., Han C.H., Leng A.J., Ma J.Y, & Liu J. (2017). Dried Rehmannia root protects against glutamate-induced cytotoxity to PC12 cells through energy metabolism-related pathways. Neural Regeneration Research, 12(8), 1338-1346.

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Western Blot Analysis of P2X7 Receptor

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Cells were detached by scraping and lysed in lysis buffer (300 μM sucrose, 1 mM K₂HPO₄, 1 mM MgSO₄, 5.5 mM glucose, 20 mM HEPES (pH 7.4), 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 0.2 μg DNase, and 0.3 μg RNase, all by Sigma-Aldrich) by repeated freeze/thawing cycles. Proteins were separated on 8% Bolt-SDS precast gels (Thermo Fisher Scientific) and blotted on nitrocellulose paper (GE Healthcare Life Science). Membranes were incubated overnight with the anti-P2×7 antibody (Sigma-Aldrich), at a dilution of 1:200 in Tris-buffered saline (TBS: 50 mM Tris, 150 mM NaCl, pH 7.6), supplemented with 3% non-fat milk and 0.5% bovine serum albumin (BSA). The anti-β actin Ab was used at a dilution of 1:1000 in TBS plus 5% BSA. The secondary anti-rabbit horseradish peroxidase-conjugated Ab (Dako, Milan, Italy) was used at a dilution of 1:2000 in TBS-t buffer (TBS, 0.1% Tween-20, Sigma-Aldrich). Protein bands were revealed with an enhanced chemiluminescence detection kit (GE Healthcare Life Science).

Amoroso F., Salaro E., Falzoni S., Chiozzi P., Giuliani A.L., Cavallesco G., Maniscalco P., Puozzo A., Bononi I., Martini F., Tognon M, & Virgilio F.D. (2016). P2×7 targeting inhibits growth of human mesothelioma. Oncotarget, 7(31), 49664-49676.

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Western Blot Analysis of GST Protein

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For western blot analysis, the protein lysate was treated with or without isopropyl β-D-1-thiogalactopyranoside, subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes. Immunoblotting was performed using an anti-GST Ab (Santa Cruz Biotechnology, Dallas, TX, USA). For visualization, a horseradish peroxidase (HRP)-conjugated secondary Ab was added, and bands were detected using an enhanced chemiluminescence detection kit (GE Healthcare Life Sciences).

Kim S.H., Cho B.H., Lee K.Y, & Jang Y.S. (2018). N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine. Immune Network, 18(3), e21.

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Retinal Protein Isolation and Western Blot

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Proteins were isolated from retinal tissues and rRECs using radioimmunoprecipitation assay buffer (Sigma-Aldrich; Thermo Fisher Scientific, Inc.). Protein concentration was determined by bicinchoninic acid kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocol. Equal amounts of protein (60 µg) were separated using SDS-PAGE on 12% gels and transferred to polyvinylidene difluoride membranes (Millipore; Merck KGaA). Following blocking with 5% non-fat milk for 1 h at room temperature, membranes were probed with rabbit anti-LC3B antibody (cat. no. ab51520; 1:3,000; Abcam) or anti-β-actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4°C. Then, membranes were washed with TBS plus Tween-20 (0.1%) and incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (cat. no. sc-516078; 1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 2 h at 37°C. Target bands were developed with an enhanced chemiluminescence detection kit (Amersham; GE Healthcare, Chicago, IL, USA). Band densities were calculated using Quantity One software version 4.5.1 (Bio-Rad Laboratories, Inc.) and normalized to β-actin.

Mao X.B., Cheng Y.H, & Xu Y.Y. (2019). miR-204-5p promotes diabetic retinopathy development via downregulation of microtubule-associated protein 1 light chain 3. Experimental and Therapeutic Medicine, 17(4), 2945-2952.

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Western Blot Analysis of Apoptotic Signaling

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Lysates were centrifuged, supernatants were collected, and protein concentration was determined using a DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). Samples were electrophoresed using 4–15% gradient polyacrylamide gels (Bio-Rad) and then transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked, rinsed and incubated with primary antibodies against unprenylated Rap1A (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), p-P38, total P38, p-Pak1, total Pak1 (Cell Signaling Technology, Inc., Danvers, MA), cleaved PARP-1 (Cell Signaling) and cleaved caspase-3 (eBioscience). After overnight incubation at 4°C, membranes were washed and incubated with their corresponding secondary antibody conjugated with horseradish peroxidase (HRP). Protein bands were detected with an enhanced chemiluminescence detection kit (GE Healthcare, Piscataway, NJ). β-Actin (Sigma Aldrich) and vinculin (Santa Cruz) were used as loading controls.

Gonzalez-Villasana V., Fuentes-Mattei E., Ivan C., Dalton H.J., Rodriguez-Aguayo C., Fernandez-de Thomas R.J., Aslan B., Monroig P.D., Velazquez-Torres G., Previs R.A., Pradeep S., Kahraman N., Wang H., Kanlikilicer P., Ozpolat B., Calin G., Sood A.K, & Lopez-Berestein G. (2015). Rac1/Pak1/p38/MMP-2 axis regulates angiogenesis in ovarian cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(9), 2127-2137.

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Tumor Tissue Protein Extraction and Analysis

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To produce the lysate from tumor tissue, 100 mg tissue was lysed and homogenized in radio immuno-precipitation Assay (RIPA) buffer (Sigma, Australia) containing 10% protease inhibitor cocktail. The samples were then centrifuged at 10,000 g at 4° C for 10 minutes and the protein content in supernatant was quantified [17 (link)]. Fifty micrograms protein were fixed on 12% gels and electrophoresed for 2 hours at 85 V and transferred to Poly vinylidene Fluoride (PVDF) membranes. The membranes were incubated with the primary antibodies ( VEGF and SPARC, 1:200 dilutions, Santa Cruz Biotechnology) for overnight at 4°C followed by one hour incubation with secondary antibodies (anti-mouse from Santa Cruz Biotechnology and anti-rabbit from Cell Signalling, USA). The bands were visualized by an enhanced chemiluminescence detection kit (GE Healthcare, Australia). The blots were then stripped using Seppro western blot stripping buffer (Sigma, Australia) and re-probed with GAPDH (1:1000 dilution, Sigma, Australia).

Noorani L., Stenzel M., Liang R., Pourgholami M.H, & Morris D.L. (2015). Albumin nanoparticles increase the anticancer efficacy of albendazole in ovarian cancer xenograft model. Journal of Nanobiotechnology, 13, 25.

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Western Blot Analysis of Myc-Tagged Proteins

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Infiltrated Nicotiana benthamiana leaves were ground in liquid nitrogen and mixed with sample buffer (100 mM Tris [pH 6.8], 20 % glycerol, 4 % SDS, and 0.2 % bromophenol blue) containing 10 % β-mercaptoethanol [43 (link)]. Samples were then boiled at 90 °C for 10 min, and centrifuged for 5 min at 13,000 × g before loading on a gel. Extracts were run in 8 % SDS-PAGE gels for the detection of AtAGO1-Myc and in 12 % SDS-PAGE gels for detecting P19-myc, P0^PL-AU-myc and P0^CLs-myc with anti-myc antibody (1:2,000; Sigma, clone 9E10), followed by an anti-mouse HRP secondary antibody (1:5,000; Bio-Rad). Antibody–protein interactions were visualized using an enhanced chemiluminescence detection kit (GE Healthcare) according to the manufacturer’s instructions.

Cascardo R.S., Arantes I.L., Silva T.F., Sachetto-Martins G., Vaslin M.F, & Corrêa R.L. (2015). Function and diversity of P0 proteins among cotton leafroll dwarf virus isolates. Virology Journal, 12, 123.

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