Clarity ecl western blotting detection reagent

Manufactured by Bio-Rad

Sourced in United States

Clarity ECL Western blotting detection reagents are a set of luminol-based chemiluminescent reagents used in Western blotting applications to detect and quantify proteins. The reagents emit light upon reaction with the enzyme-labeled secondary antibody, allowing for the visualization and analysis of target proteins.

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Lab products found in correlation

Immobilon p, by Merck Group (3 mentions) Anti gfp primary antibody, by Merck Group (2 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions) Horseradish peroxidase conjugated anti rabbit or anti mouse secondary antibody, by Cell Signaling Technology (2 mentions) Nitrocellulose, by Thermo Fisher Scientific (1 mentions) Molecular imager chemidoc xrs, by Bio-Rad (1 mentions) Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions) Coomassie protein assay reagent, by Merck Group (1 mentions) Bis tris gel, by Thermo Fisher Scientific (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Gadph antibody, by Cell Signaling Technology (1 mentions) X ray film, by Bio-Rad (1 mentions) Bcl 2, by Cell Signaling Technology (1 mentions) Bradford assay, by Bio-Rad (1 mentions) Phosphatase inhibitor cocktail, by Roche (1 mentions) β actin antibody, by Cell Signaling Technology (1 mentions) X ray film, by Carestream (1 mentions)

7 protocols using clarity ecl western blotting detection reagent

Extraction and Analysis of A. fumigatus Proteins

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To extract proteins from A. fumigatus mycelia, conidia from related strains were incubated in liquid-inducing medium and then shaken at 220 rpm on a rotary shaker at 37 °C for 48 h. The mycelium was ground in liquid nitrogen using a mortar and pestle and suspended in ice-cold extraction buffer (50 mM HEPES (pH 7.4), 137 mM KCl, 10% glycerol, 1 mM EDTA, 1 μg/mL pepstatin A, 1 μg/mL leupeptin, and 1 mM PMSF). Equal amounts of proteins (40 μg) per lane were subjected to 10% SDS – PAGE and transferred to PVDF membranes (Immobilon-P, Millipore) in 384 mM glycine, 50 mM Tris (pH 8.4), and 20% methanol at 250 mA for 1.5 h, the membranes were blocked with PBS, 5% milk, and 0.1% Tween 20. Next, the membrane was probed sequentially with 1:3000 dilutions of anti-GFP primary antibody (Sigma) and goat anti-rabbit IgG-horseradish peroxidase secondary antibody (Abclonal, AS014) diluted in PBS, 5% milk, and 0.1% Tween 20. Blots were developed using Clarity ECL Western blotting detection reagents (Bio-Rad), and images were acquired with a Tanon 4200 Chemiluminescence Imaging System.

Li X., Feng R., Luo P., Zhang Y, & Lu L. (2023). Synergistic effects of putative Ca2+-binding sites of calmodulin in fungal development, temperature stress and virulence of Aspergillus fumigatus. Virulence, 15(1), 2290757.

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Protein extraction and Western blotting

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2×10⁶ T cells were incubated for 45min in ice-cold lysis buffer (10mM Tris, 50mM NaCl, 5mM EDTA and 1% Triton X-100) containing various protease and phosphatase inhibitors (50mM NaF, 1mM Na₃VO₄, 30mM sodium pyrophosphate, 1mM PMSF, 2µg/ml leupeptin, 2µg/ml aprotinin). Lysates were then centrifuged at 12.000 g for 15min at 4°C to remove insoluble material. Protein concentration was determined using the Coomassie protein assay reagent (Sigma Aldrich). Proteins (20µg per lane) were separated in 4 to12% gradient (wt/vol) Bis-Tris gels (Life Technologies) and transferred to nitrocellulose (Life Technologies) or PVDF (Millipore) membrane. Membranes were blocked for 1h with Tris-buffered saline solution containing 0.05% Tween (TBS-T) and 5% non-fat dry milk at room temperature. Following blocking, membranes were incubated overnight at 4°C with the indicated antibody in blocking solution on a shaking surface and were then washed and incubated with the appropriate secondary HRP-conjugated antibody for 1.5h at room temperature. Detection was performed with the Clarity ECL Western Blotting detection reagents (Biorad) and membranes were visualized by the ChemiDoc XRS⁺ Molecular Imager (Biorad). Densitometric analysis was performed using the ImageJ software and results are expressed as densitometric ratios of tyrosine phosphorylated proteins or SAP over β-actin ± SEM.

Karampetsou M.P., Comte D., Kis-Toth K., Terhorst C., Kyttaris V.C, & Tsokos G.C. (2016). Decreased SAP expression in T cells from patients with SLE contributes to early signaling abnormalities and reduced IL-2 production. Journal of immunology (Baltimore, Md. : 1950), 196(12), 4915-4924.

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Protein Extraction from A. fumigatus Mycelia

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To extract proteins from A. fumigatus mycelia, conidia from related strains were incubated in liquid-inducing medium and then shaken at 220 rpm on a rotary shaker at 37 °C for 48 h. The mycelium was ground in liquid nitrogen with a mortar and pestle and suspended in an ice-cold extraction buffer (50 mM HEPES pH 7.4, 137 mM KCl, 10% glycerol, 1 mM EDTA, 1 μg/mL pepstatin A, 1 μg/mL leupeptin, 1 mM PMSF). Equal amounts of proteins (40 μg) per lane were subjected to 10% SDS–PAGE and transferred to PVDF membranes (Immobilon-P, Millipore) in 384 mM glycine, 50 mM Tris (pH 8.4), and 20% methanol at 250 mA for 1.5 h, and the membranes were then blocked with phosphate-buffered saline (PBS), 5% milk, and 0.1% Tween 20. Next, the membrane was probed sequentially with 1:3000 dilutions of an anti-GFP primary antibody (Sigma) and goat anti-rabbit IgG-horseradish peroxidase secondary antibody (Abclonal Co., AS014, Woburn, MA, USA) diluted in PBS, 5% milk, and 0.1% Tween 20. Blots were developed using Clarity ECL western blotting detection reagents (Bio-Rad, Hercules, CA, USA), and images were acquired with a Tanon 4200 Chemiluminescence Imaging System (Tanon, St Andrews, Scotland).

Sun C., Li X., Zhang Y, & Lu L. (2022). Subunit C of V-ATPase-VmaC Is Required for Hyphal Growth and Conidiation in A. fumigatus by Affecting Vacuolar Calcium Homeostasis and Cell Wall Integration. Journal of Fungi, 8(11), 1219.

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Western Blot Analysis of GFP-tagged Aspergillus

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Western blotting was carried out as previously reported (51 (link)). Briefly, 1 × 10⁶ conidia of the AfERG10A::GFP and WT strains were inoculated in liquid CM and shaken at 37°C for 48 h. The mycelia were harvested and ground in liquid nitrogen with a mortar and pestle and suspended in ice-cold extraction buffer (50 mM HEPES [pH 7.4], 137 mM KCl, 10% glycerol, 1 mM EDTA, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride [PMSF]). Ten micrograms of proteins per lane was loaded onto a 10% SDS-PAGE gel. After electrophoresis, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore) in 384 mM glycine, 50 mM Tris (pH 8.4), and 20% methanol at 250 mA for 1.5 h. The membrane was then blocked with phosphate-buffered saline (PBS) containing 5% milk and 0.1% Tween 20. The membrane was then incubated in anti-mouse GFP primary antibody (Roche) at a 1:20,000 dilution and goat anti-mouse IgG-horseradish peroxidase secondary antibody at a 1:5,000 dilution. Blots were developed using the Clarity ECL Western blotting detection reagents (Bio-Rad), and images were acquired with a Tanon 4200 chemiluminescent imaging system (Tanon).

Zhang Y., Wei W., Fan J., Jin C., Lu L, & Fang W. (2020). Aspergillus fumigatus Mitochondrial Acetyl Coenzyme A Acetyltransferase as an Antifungal Target. Applied and Environmental Microbiology, 86(7), e02986-19.

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Extracting Proteins from Aspergillus nidulans

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To extract proteins from A. nidulans mycelia, conidia from alcA(p)::GFP-akrA and the wild-type strains were inoculated in the liquid inducing medium, then shaken at 220 rpm on a rotary shaker at 37°C for 24 h. The mycelium was ground in liquid nitrogen with a mortar and pestle and suspended in ice-cold extraction buffer (50 mM HEPES pH 7.4, 137 mM KCl, 10% glycerol containing, 1 mM EDTA, 1 μg/mL pepstatin A, 1 μg/mL leupeptin, 1 mM PMSF). Equal amounts of protein (40 μg) per lane were subjected to 10% SDS–PAGE, transferred to PVDF membrane (Immobilon-P, Millipore) in 384 mM glycine, 50 mM Tris (pH 8.4), 20% methanol at 250 mA for 1.5 h, and the membrane was then blocked with PBS, 5% milk, 0.1% Tween 20. Next, the membrane was then probed sequentially with 1:3000 dilutions of the primary antibodies anti-GFP or anti-FLAG or anti-actin and goat anti-rabbit IgG-horseradish peroxidase diluted in PBS, 5% milk, 0.1% Tween 20. Blots were developed using the Clarity ECL Western blotting detection reagents (Bio-Rad), and images were acquired with the Tanon 4200 Chemiluminescent Imaging System (Tanon).

Zhang Y., Zheng Q., Sun C., Song J., Gao L., Zhang S., Muñoz A., Read N.D, & Lu L. (2016). Palmitoylation of the Cysteine Residue in the DHHC Motif of a Palmitoyl Transferase Mediates Ca2+ Homeostasis in Aspergillus. PLoS Genetics, 12(4), e1005977.

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Western Blot Analysis of Apoptosis Markers

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Total cell lysates were collected by lysing H9C2 cardiomyocytes with lysis buffer supplemented with Protease Inhibitor Cocktail (Thermo Fisher Scientific) and Phosphatase Inhibitor Cocktail (Roche). The protein concentration of the cell lysate was measured by Bradford assay (Bio-Rad, CA, USA). The extracted protein samples were separated by 8%–12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes for detection with appropriate antibodies. Primary antibodies against total caspase 3 (1 : 1000), cleaved caspase 3 (1 : 1000), Bax (1 : 1000), Bcl-2 (1 : 1000), GADPH antibody (1 : 1000), and horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (1 : 3000) were purchased from Cell Signaling Technology. Anti-Ptpn4 antibody (1 : 1000) was purchased from Novus. Blots were visualized with Clarity ECL Western Blotting Detection Reagent (Bio-Rad) and subsequently exposed to X-ray film (Carestream, NY, USA). ImageJ software (National Institutes of Health, MD, USA) was used to analyze the optical densities of the immunoreactive bands. Protein expression was normalized to that of loading control (GAPDH).

Ge L., Cai Y., Ying F., Liu H., Zhang D., He Y., Pang L., Yan D., Xu A., Ma H, & Xia Z. (2019). miR-181c-5p Exacerbates Hypoxia/Reoxygenation-Induced Cardiomyocyte Apoptosis via Targeting PTPN4. Oxidative Medicine and Cellular Longevity, 2019, 1957920.

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Western Blot Analysis of Autophagy and Inflammasome Markers

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H9c2 cardiomyocytes were lysed with lysis buffer supplemented with Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail, and the total cell lysate was collected. The protein concentration of the cell lysate was determined by Bradford assay. The extracted protein samples were separated by 8–12.5% 12 alkyl sulphate polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membrane, and detected with appropriate antibodies. Primary antibodies against β‐actin antibody (1: 1,000), total LC3 (1:1,000), p62 (1:1,000), NLRP3 (1:1,000) and horseradish peroxidase‐conjugated anti‐rabbit or anti‐mouse secondary antibodies (1: 3,000) were purchased from Cell Signaling Technology. IL‐1, IL‐18 and ASC (1:1,000) were purchased from Abcam (Cambridge, MA, USA), and caspase‐1p20 antibody (1:1,000) was purchased from Santa Cruz (Santa Cruz, CA, USA). Blotted polyvinylidene difluoride membranes were incubated by Clarity ECL Western Blotting Detection Reagent (Bio‐Rad, Hercules, CA, USA) and exposed to X‐ray film (Carestream, Rochester, NY, USA). ImageJ (National Institutes of Health, Bethesda, MA, USA) was used to analyze the optical densities of the immunoreactive bands. β‐Actin expression was used as the loading control.

Zhang D., He Y., Ye X., Cai Y., Xu J., Zhang L., Li M., Liu H., Wang S, & Xia Z. (2020). Activation of autophagy inhibits nucleotide‐binding oligomerization domain‐like receptor protein 3 inflammasome activation and attenuates myocardial ischemia‐reperfusion injury in diabetic rats. Journal of Diabetes Investigation, 11(5), 1126-1136.

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