Samples were separated by electrophoresis in 4 to 20% SDS–polyacrylamide gel electrophoresis gels with 30 μg per lane, transferred to a polyvinylidene difluoride membrane, and blotted with antibodies. Secondary horseradish peroxidase (HRP)–conjugated secondary antibodies (Santa Cruz Biotechnology) and Amersham ECL Prime Western Blotting Detection Reagents (catalog no. RPN2232, GE Healthcare) were used for detection. For high–molecular weight proteins, samples were separated by electrophoresis in 3 to 18% tris-acetate gel electrophoresis gels with 30 μg per lane, transferred to a polyvinylidene difluoride membrane, and blotted with antibodies. Secondary HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) and Amersham ECL Prime Western Blotting Detection Reagents (catalog no. RPN2232, GE Healthcare) were used for detection.
Amersham ecl prime western blotting detection reagent
Manufactured by GE Healthcare
Sourced in United Kingdom, United States, Germany, Japan, Sweden, France, New Zealand, China, Canada
The Amersham ECL Prime Western Blotting Detection Reagent is a chemiluminescent substrate used for the detection of proteins in Western blotting analysis. It provides a sensitive and quantitative way to detect and analyze protein expression levels.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (52 mentions)
Ripa buffer, by Thermo Fisher Scientific (33 mentions)
Pvdf membrane, by Merck Group (30 mentions)
Chemidoc mp imaging system, by Bio-Rad (23 mentions)
Amersham imager 600, by GE Healthcare (20 mentions)
Trans blot turbo transfer system, by Bio-Rad (14 mentions)
β actin, by Cell Signaling Technology (14 mentions)
Ripa buffer, by Merck Group (14 mentions)
Imagequant las 4000, by GE Healthcare (13 mentions)
Chemidoc touch imaging system, by Bio-Rad (12 mentions)
Protease inhibitor cocktail, by Merck Group (11 mentions)
Nitrocellulose membrane, by Bio-Rad (11 mentions)
Pvdf membrane, by Bio-Rad (11 mentions)
Image lab software, by Bio-Rad (10 mentions)
Protease inhibitor cocktail, by Roche (10 mentions)
Chemidoc xrs system, by Bio-Rad (10 mentions)
Immobilon p, by Merck Group (10 mentions)
Protease inhibitor, by Roche (9 mentions)
Laemmli sample buffer, by Bio-Rad (9 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (9 mentions)
489 protocols using amersham ecl prime western blotting detection reagent
1
Quantitative Western Blot Analysis
2
Protein Secretions Analysis by Western Blot
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Western blot analysis was carried out to check the secretions of the individual BCSP31, Omp3b, and SOD proteins from HJL228, HJL219, and HJL213, respectively, using the modified method mentioned in previous study (Hur and Lee, 2011a (link)). Briefly, the proteins in supernatant of culture were separated by 12% SDS-PAGE gel and transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The individual proteins were reacted with anti-His antibodies (Invitrogen, Grand Island, NY, USA) and horseradish peroxidase-conjugated rabbit anti-mouse IgG antibodies (Southern Biotech., Birmingham, AL, USA). Immunoreactive bands were detected by addition of the AmershamTM ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and the CheBi illumination system (Neo science, Suwon, Gyeonggi, South Korea).
3
mRNA m6A Methylation Analysis
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Total RNA was isolated using RNAiso Plus (Total RNA extraction reagent, Takara, 9109) according to the manufacturers’ instructions and quantified by fluorometry. Poly(A)+ RNA from total RNA was isolated using the GenEluteTM mRNA Miniprep Kit (Sigma, MRN70-1KT) according to the manufacturers’ instructions and quantified by QubitTM RNA HS Assay Kit (Invitrogen, Q32855). The Poly(A)+ RNA samples were loaded to the AmershamTM Hybond-N + membrane (GE Healthcare, RPN119B) with a Bio-Dot Apparatus (Bio-Rad, 170-6545) and UV-crosslinked to the membrane. Then the membrane was blocked with 5% non-fat dry milk (in 1 × Tris-buffered saline with 0.1% Tween® 20 detergent) for 1–2 h and incubated with a specific anti-m6A antibody (Synaptic Systems, 202003, 1:2000) overnight at 4 °C. Then the membrane was further incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cell signaling Technology, 7074S, 1:5000) for 1 h at room temperature. The membrane was developed with AmershamTM ECL Prime Western Blotting Detection Reagent (GE Healthcare, RPN2232). To ensure equal spotting of mRNA onto the membrane, the same blot was stained with 0.02% methylene blue in 0.3 M sodium acetate.
4
Western Blot Analysis of Jurkat Cells
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Samples of Jurkat cells lysates were prepared as previously described [8 (link)]. Equal amounts of proteins (50 μg) were separated by SDS–PAGE on 10% polyacrylamide gels and transferred to nitrocellulose membranes. Nonspecific binding sites were blocked with blocking buffer (5% nonfat dried milk in PBS 0.1% Tween 20). Membranes were incubated overnight at 4° C with a primary antibody CYP3A4, pStat1-Y701, Stat1, GAPDH and β-tubulin (Santa Cruz) pNF-κB-S536 (Cell Signaling). Secondary mouse or rabbit HRP-conjugated antibody (Santa Cruz Biotechnology) were incubated for 1h at room temperature. AmershamTM ECL™ Prime Western blotting detection reagent (GE Healthcare) was used to develop the protein blot. Blots were developed using ImageQuant LAS 4000 (GE Health Care). Densitometry analysis was performed by Image J (version 5.1, Silk Scientific Corporation, NIH, Bethesda, MA) software. Experimental values were referred to those obtained with the corresponding loading protein band.
5
Quantifying Oxidized Peroxiredoxins in Zebrafish
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Isolation and quantification of total cellular protein as well as Hif-1α western blots were performed according to standard procedures described previously [10] . For quantification of peroxiredoxins, membranes were incubated with Prx-SO 2/3 antibody (ab16830, Abcam, UK) 1:10, 000 in 5% [w/v] bovine serum albumin (BSA) overnight at 4°C, after blocking with 5% [w/v] milk. Binding of the primary antibody was detected with a secondary antibody conjugated to horseradish peroxidase (HRP; Sigma-Aldrich, Germany; diluted 1:1000 in blocking buffer, incubated for 1h at room temperature) using the Amersham TM ECL Prime Western Blotting Detection Reagent (GE Healthcare, UK). Semiquantification of oxidized peroxiredoxins was accomplished by using the Image Lab TM software (Bio-Rad, Germany). The responsiveness of Zebrafish Prx ox protein levels to increasing concentrations of H 2 O 2 in the medium is shown in (Fig. 10,C).
6
Histone Modification Analysis in CD11b+ Cells
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CD11b+ cells from PBS injected or WGP-injected mice (24 h) were separated using CD11b microbeads (Miltenyi Biotech). Histone was extracted according to the manufacturer’s instruction (Active Motif, Inc.) and protein concentration was determined using Bradford quantification. Histone proteins were separated by SDS-PAGE 15% Tris-HCl gels and transferred onto PVDF membranes (Millipore). The membrane was blocked with 5% BSA at room temperature for 1 h and incubated overnight at 4 °C with primary antibodies including tri-methyl-histone H3 (lys4) rabbit mAb, acetyl-histone H3 (lys27) XP Rabbit mAb, tri-methyl-histone H3 (Lys27) Rabbit mAb, and histone H3 XP rabbit mAb (Cell Signaling Technology), then incubated with HRP-conjugated secondary antibodies (GE Healthcare) at room temperature for 1 h. The membrane was developed with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare) and detected through Medical Film Processor (Konica Minolta Medical & Graphic). Precision Plus Protein Kaleidoscope Prestained Protein Standards were used as a standard protein marker (Bio-Rad).
7
Western Blot Analysis of Myelin and Cytoskeletal Proteins
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Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 4% to 20% gradient gel (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred onto polyvinylidene fluoride or nitrocellulose membranes (Thermo Fisher Scientific). Membranes were blocked either with 5% bovine serum albumin (BSA, Sigma-Aldrich) or 5% milk in Tris-buffered saline (TBS). Proteins were analyzed with rabbit antibodies against myelin basic protein (Mbp, 1:400, AB980, Thermo Fisher Scientific), microtubule-associated protein 2 (Map2, 1:500, ab24640, Abcam, Cambridge, UK) and β-Tubulin (1:4000, ab6046, Abcam) in 5% milk (for Mbp) or 5% BSA (for Map2 and β-Tubulin) in TBS-Tween 20 (TBS-T, Sigma-Aldrich) overnight at 4 °C. Horseradish peroxidase-coupled donkey anti-rabbit (1:1000, GE Healthcare Life Science, Piscataway, NJ, USA) antibody was used as a secondary antibody and incubated with the membranes for 1 h at room temperature. Binding was detected using the chemiluminescent Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) on a Chemidoc XRS+ system (Bio-Rad). Pixel summation of individual bands was performed with ImageJ Software (NIH, Bethesda, MD, USA). The ratio between Mbp/Map2 and β-Tubulin was calculated for each animal.
8
Quantifying PDK4 Protein Levels in Lung Tissue
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Western blot analyses were performed to evaluate PDK4 protein levels. Lung tissues excised from neonatal mice (<12 h old, n = 12) and adult male mice (8 weeks old, n = 12) exposed to normoxia or hyperoxia for 96 h were homogenized in tissue extraction buffer (78510; Thermo Fisher Scientific, Waltham, MA, USA). The lysates were clarified by centrifugation at 10,000 g for 5 min. Protein samples (50 μg) were separated using SDS/PAGE and transferred to membranes. The membranes were blocked and incubated with the anti-PDK4 primary antibody (1:500; ab172920; Abcam) or anti-β-actin antibody (1:5000; GTX26276; GeneTex, Irvine, CA, USA), followed by horseradish peroxidase (HRP)-labeled secondary antibodies. Chemiluminescence was detected using the Amersham ECL Prime Western blotting detection reagent (GE Healthcare, Chicago, IL, USA) with a digital imaging system (Bio-Rad ChemiDoc XRS+; Bio-Rad Laboratories, Hercules, CA, USA).
9
SDS-PAGE and Western Blot Analysis
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Aliquots were withdrawn from cultures at OD600 of 0.3 and 0.5, spun down, and pellets resuspended in 1× loading dye [4× Laemmli sample buffer (Bio‐Rad), 1/10 volume of β‐mercaptoethanol added before use]. Samples were incubated for 5 min at 95°C before gel electrophoresis. Proteins were separated on an Any kD™ Mini‐PROTEAN® TGX Stain‐Free™ Gel (Bio‐Rad), followed by fluorescent detection of total protein by ChemiDoc™ MP (Bio‐Rad) and the “Any kD™ Mini‐PROTEAN® TGX Stain‐Free™ Gel” application. Then, proteins were transferred to a PVDF membrane with the Trans‐Blot Turbo™ Mini PVDF transfer Packs (Bio‐Rad) and the Trans‐Blot Turbo Transfer System (Bio‐Rad) using the “Any Kd” preset. After o/n blocking in Odyssey® Blocking Buffer (PBS; LI‐COR) at 4°C, membranes were incubated for 1 h with monoclonal ANTI‐FLAG M2‐Peroxidase (HRP) mouse antibody (Sigma; cat. nr. A8592) at r.t., followed by three washes with PBS‐T, and two with PBS. Blots were developed with Amersham™ ECL™ Prime Western Blotting Detection Reagent (GE Healthcare) and imaged using ChemiDoc™ MP (Bio‐Rad) with Chemi Hi Resolution application. Images were analyzed with Image Lab Software (version 4.0 build 16); the quantification of band intensities in Fig 6 used equal‐sized squares and pixel counts for background, DgcM, and OmpA.
10
Protein Quantification and Western Blot
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Equal amounts of proteins (20 μg per sample) from clarified lysates were loaded onto SDS-PAGE gels. After separation, the proteins were transferred by using the eBlot protein transfer system (GenScript) on polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 5% bovine serum albumin (BSA; Calbiochem) in phosphate-buffered saline (PBS) with 0.1% Tween 20 for 1 h before overnight incubation with primary antibodies. After washes in PBS–0.1% Tween 20, the membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology). The signal was then developed with the Amersham ECL Prime Western blotting detection reagent (GE Healthcare), and the images were acquired with a LAS-3000 luminescent image analyzer (Fujifilm).
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