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Amersham ecl western blotting detection system

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The Amersham ECL western blotting detection system is a laboratory equipment product designed for the detection and analysis of proteins in Western blot assays. It utilizes a chemiluminescent detection method to visualize and quantify target proteins.

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Lab products found in correlation

Amersham ecl western blotting, by Cytiva (7 mentions) Bradford protein assay, by Bio-Rad (2 mentions) Hybond ecl nitrocellulose membrane, by GE Healthcare (2 mentions) Ripa buffer, by Cell Signaling Technology (2 mentions) Blotto a, by Santa Cruz Biotechnology (2 mentions) Hybond, by Cytiva (2 mentions) Monoclonal anti β actin, by Merck Group (1 mentions) Bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions) Lysis buffer, by Cell Signaling Technology (1 mentions) Emr2 antibody, by Abcam (1 mentions) Horseradish peroxidase, by Cell Signaling Technology (1 mentions) Vascular endothelial growth factor (vegf), by Abcam (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Bca protein assay, by Thermo Fisher Scientific (1 mentions) Bca protein assay kit, by Bio-Rad (1 mentions) Pierce protease and phosphatase inhibitor mini tablet, by Thermo Fisher Scientific (1 mentions) Nitrocellulose blotting membrane, by GE Healthcare (1 mentions)

Close spelling variants of this product

ECL system (381 citations) ECL detection system (314 citations) Amersham ECL (119 citations) Amersham ECL system (12 citations) Western blotting detection system (10 citations) AmershamTM ECLTM (3 citations)

7 protocols using amersham ecl western blotting detection system

1

Quantitative Western Blot Analysis

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Whole proteins were extracted in RIPA buffer (Cell Signaling) and quantified by the Bradford protein assay (Biorad). Samples were separated by 12% SDS–PAGE and transferred to Hybond ECL nitrocellulose membranes (GE Healthcare). The membranes were blocked with Blotto A (Santa Cruz) at room temperature for 1 h, and incubated with the appropriate primary antibodies for 2 h at room temperature, as previously described (Vergara et al., 2016 (link)). After two washes with a solution of TBS containing 0.1% (v/v) tween 20 for 10 min, the membranes were incubated with secondary antibody HRP-conjugated for 2 h at room temperature (standard dilution 1:2,000). Blots were then developed using the Amersham ECL western blotting detection system (GE Healthcare). Densitometric quantitation of at least three independent replicates was done using ImageJ software.
Vergara D., Stanca E., Guerra F., Priore P., Gaballo A., Franck J., Simeone P., Trerotola M., De Domenico S., Fournier I., Bucci C., Salzet M., Giudetti A.M, & Maffia M. (2017). β-Catenin Knockdown Affects Mitochondrial Biogenesis and Lipid Metabolism in Breast Cancer Cells. Frontiers in Physiology, 8, 544.
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2

Pro-IL-1β Expression Analysis in AGT3 and rhGGT Treated Cells

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The cells were pretreated with or without AGT3 (100 ng/ml) for 2 h followed by 100ng/ml of rhGGT, and then cells were harvested 1day or 2 d after rhGGT stimulation. Western blotting was carried out as previous description [9 (link)]. Cell pellets were resuspended in ice-cold lysis buffer as described previously. Proteins were separated by SDS-PAGE and electro-blotted onto nitrocellulose membrane. The membrane was blocked by 3% milk for 30min. Pro-IL-1β was detected by polyclonal IL-1β antibody (1:500; Santa Cruz Biothechnology, Inc. Texas, USA) and anti-rabbit secondary antibody. Monoclonal anti-β-actin (1:10000, Sigma-Aldrich Co., MI, USA) was used as internal control. The results were visualized by Amersham ECL western blotting detection system (GE Healthcare, Japan).
Kawazoe Y., Miyauchi M., Nagasaki A., Furusho H., Yanagisawa S., Chanbora C., Inubushi T., Hyogo H., Nakamoto T., Suzuki K., Moriwaki S., Tazuma S., Niida S, & Takata T. (2015). Osteodystrophy in Cholestatic Liver Diseases Is Attenuated by Anti-γ-Glutamyl Transpeptidase Antibody. PLoS ONE, 10(9), e0139620.
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3

Western Blot Protein Analysis

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Cell lysates were generated using 10x lysis buffer (Cell-Signaling) containing 20 mM Tris–HCl(pH7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1 % Triton, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, 1 mM Na3VO4, and 1 μg/ml leupeptin. Protease and phosphatase inhibitor (Roche) and 1 mM phenylmethasnesulfonylfluoride were added. Lysates were cleared of insoluble materials by centrifugation. Protein concentrations were normalized by bicinchoninic acid assay (Thermo Scientific) to 1 μg/μl in 1XSDS loading buffer. Protein samples were heated to 90 °C for 5 min then electrophoresed through a 4–20 % Tris–Glycine(Invitrogen). Resolved proteins were then transferred to an Immun-Blot PVDF membrane, blocked in 5 % milk in Tris-buffered saline and incubated in primary antibody overnight at 4 °C. EMR2 antibody was applied at 1:500(Abcam) and anti-GAPDH antibody at 1:10,000 (cell-vignaling). After incubation a secondary antibody was conjugated to horseradish peroxidase (cell-signaling) diluted to 1:2,000 in 5 % milk. Membranes were developed using the Amersham ECL Western blotting detection system (GE Healthcare).
Ivan M.E., Safaee M., Oh T., Clark A.J., Sun M.Z., Kim J., Bloch O., Jahangiri A., Phillips J.J., Aghi M.K, & Parsa A.T. (2014). Epidermal growth factor-like module containing mucin-like hormone receptor 2 expression in gliomas. Journal of neuro-oncology, 121(1), 53-61.
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4

Western Blot Analysis of EGFR and VEGF

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Cells were grown to 70%–80% confluence, washed twice with cold PBS, and lysed [lysis buffer: 50mM Tris-HCl (pH7.5), 150mM NaCl, 1% (v/v) NP40, 0.5% (w/v) sodium deoxycholate, 1mM EDTA, 0.1% SDS and a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN)]. Lysates were collected by centrifugation at 12000 × g for 20 mins at 40°C. BCA protein assay (Thermo Scientific, Rockford, IL) measured protein concentrations. Lysates with 10 µg of total protein were resolved by SDS PAGE and transferred to PVDF membranes. Membranes were incubated with the primary antibody (EGFR, Abcam, #15669 and VEGF, Abcam, #46154), washed, incubated with horseradish peroxidase conjugated secondary antibodies, washed again and detected by the Amersham ECL Western blotting detection system (GE healthcare, Buckinghamshire, UK). Membranes were reprobed with horseradish peroxidase-conjugated mouse monoclonal antihuman beta-actin.
Sweeny L., Prince A., Patel N., Moore L.S., Rosenthal E.L., Hughley B.B, & Warram J.M. (2016). Antiangiogenic Antibody Improves Melanoma Detection by Fluorescently Labeled Therapeutic Antibodies. The Laryngoscope, 126(12), E387-E395.
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5

Western Blot Protein Analysis Protocol

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Cells were harvested and washed with PBS, and then lysed with lysis buffer supplemented with Pierce Protease and Phosphatase Inhibitor Mini Tablets (ThermoFisher Scientific) for 10 min on ice. The whole cell lysate was scraped from the plates and then centrifuged at 12000 rpm for 10 min at 4 °C. The concentrations of protein were determined by BCA Protein Assay Kit (Bio-Rad). Cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose blotting membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA), then incubated with primary specific antibodies at 4 °C overnight. The membranes were incubated with secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) at 1:10000 dilution for 2 h at room temperature. The protein band intensities were evaluated using Amersham ECL Western Blotting Detection System (GE Healthcare Life Sciences) and were normalized to housekeeping genes, either β-actin or HSP90. All western blot experiments were performed at least three times.
Gao P., Wang X., Jin Y., Hu W., Duan Y., Shi A., Du Y., Song D., Yang M., Li S., Han B., Zhao G., Zhang H., Fan Z, & Miao Q.R. (2018). Nogo-B receptor increases the resistance to tamoxifen in estrogen receptor-positive breast cancer cells. Breast Cancer Research : BCR, 20, 112.
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6

Protein Extraction and Western Blot Analysis

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Protein samples were extracted from liver tissue or cultured AML-12 cells with procedures as described in detail elsewhere [38 ]. For insoluble fraction preparation, pellets were washed with RIPA and resuspended in 2% SDS Tris-buffer. Protein concentrations were determined using a BCA reagent. Equal amounts of protein were subject to SDS-PAGE. Binding of the primary antibody was detected by peroxidase-conjugated secondary antibodies and enhanced chemiluminescence, and bands were quantified with Amersham ECL Western Blotting Detection System (GE Healthcare).
Zhang X.W., Mi S., Li Z., Zhou J.C., Xie J., Hua F., Li K., Cui B., Lv X.X., Yu J.J, & Hu Z.W. (2016). Antagonism of Interleukin-17A ameliorates experimental hepatic fibrosis by restoring the IL-10/STAT3-suppressed autophagy in hepatocytes. Oncotarget, 8(6), 9922-9934.
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7

Quantitative Western Blot Analysis

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Whole proteins were extracted in RIPA buffer (Cell Signaling, Danvers, MA) and quantified by the Bradford protein assay (BIORAD, Hercules, CA). Samples were separated by 12% SDS-PAGE and transferred to Hybond ECL nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). The membranes were blocked with Blotto A (Santa Cruz, Dallas, Texas) at room temperature for 1 h, and incubated with the appropriate primary antibodies (see Supplementary Table 1) for 2 h at room temperature, as previously performed. 20 After two washes with a solution of TBS containing 0.1% (v/v) tween 20 for 10 min, the membranes were incubated with secondary antibody HRPconjugated for 2 h at room temperature (standard dilution 1:2000). Blots were then developed using the Amersham ECL western blotting detection system (GE Healthcare, Little Chalfont, UK).
Densitometric quantitation of at least three indipendent replicates was done using ImageJ software.
Vergara D., De Domenico S., Tinelli A., Stanca E., Del Mercato L.L., Giudetti A.M., Simeone P., Guazzelli N., Lessi M., Manzini C., Santino A., Bellina F, & Maffia M. (2017). Anticancer effects of novel resveratrol analogues on human ovarian cancer cells. Molecular bioSystems, 13(6).
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