Ecl western blotting substrate

Manufactured by GE Healthcare

Sourced in United Kingdom, United States, Germany

The ECL Western blotting substrate is a chemiluminescent detection system used in Western blot analysis to visualize and quantify target proteins. It utilizes a horseradish peroxidase-catalyzed reaction to produce a luminescent signal that can be detected and measured.

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Lab products found in correlation

Vinculin, by Cell Signaling Technology (3 mentions) Bca assay, by Merck Group (2 mentions) γ h2ax, by Abcam (2 mentions) Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (2 mentions) β actin, by Merck Group (2 mentions) Ripa buffer, by Merck Group (2 mentions) Pvdf membrane, by GE Healthcare (2 mentions) Chemidoc xrs system, by Bio-Rad (2 mentions) Chemidoc, by Bio-Rad (2 mentions) Chemocam device, by Intas (2 mentions) Cyclin d1, by Cell Signaling Technology (1 mentions) Cyclin e1, by Cell Signaling Technology (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Ab15597, by Abcam (1 mentions) Hmox1, by Abcam (1 mentions) Keap1 10503 2 ap, by Proteintech (1 mentions) Ab13243, by Abcam (1 mentions) Protease and phosphatase inhibitor tablet, by Roche (1 mentions) Odyssey fc, by LI COR (1 mentions) Image studio software, by LI COR (1 mentions)

Close spelling variants of this product

ECL substrate (81 citations) ECL Western Blotting Substrate kit (7 citations) ECL Prime Western Blotting substrate (6 citations) Pierce ECL Western Blotting Substrate (6 citations) Amersham ECL Western blotting substrate (5 citations) ECL start Western Blotting Substrate (3 citations) ECL™ Plus Western Blotting System (Immobilon Western Chemiluminescent HRP substrate; (2 citations)

26 protocols using ecl western blotting substrate

Western Blot Analysis of PARP and Cell Cycle Markers

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W780 and W0069 cells treated with Talazoparib or NanoTLZ were lysed in RIPA buffer (1 M Tris-Cl, 5 M NaCl, pH 7.4, 0.5 M EDTA, 25 mM deoxycholic acid, 1% triton-X, 0.1% SDS) with protease inhibitors (1 mM PMSF, 2 µg/ml aprotinin and 5 µg/ml leupeptin). Tumor samples dissected from BRCA-deficient mice were homogenized and lysed in EBC buffer (5 mol/L NaCl, 1 mol/L Tris pH 8) with protease inhibitors and 10% NP-40. Protein concentrations were determined using the BCA assay (Sigma-Aldrich). 20 µg of protein were separated by 10% SDS-PAGE gels and transferred to nitrocellulose membranes. γH₂AX (Abcam, 1:1000), cleaved-caspase 3 (Cell Signaling, 1:1000), PARP/cleaved-PARP (Cell Signaling, 1:1000), Cyclin D1 (Cell Signaling, 1:1000), Cyclin E1 (Cell Signaling, 1:1000), PCNA (Santa Cruz, 1:1000), and vinculin (Cell Signaling, 1:4000) primary antibodies were used to detect the corresponding proteins. Secondary antibodies (anti-rabbit or anti-mouse linked to HRP) were purchased from Cell Signaling. ECL Western blotting substrate (GE Healthcare Life Sciences, UK) was used to detect the signal. Images shown are representative of 3 independent experiments. ImageJ was used to quantify protein expression.

Zhang D., Baldwin P., Leal A.S., Carapellucci S., Sridhar S, & Liby K.T. (2019). A nano-liposome formulation of the PARP inhibitor Talazoparib enhances treatment efficacy and modulates immune cell populations in mammary tumors of BRCA-deficient mice. Theranostics, 9(21), 6224-6238.

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Western Blot Analysis of Cellular Proteins

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Cell lysates were prepared by whole cell extract buffer (20 mM Tris pH 7.5, 400 mM NaCl, 10% Glycerol, 0.5% NP40, 0.1% SDS) and were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4%−12% Bis-Tris gel) and transferred to PVDF membrane or nitrocellulose (Figure 2). Protein concentrations were determined by Bradford or BCA protein assay reagent (Pierce, Rockford, IL). The PVDF membrane was incubated in a blocking buffer for 1 hour and then with a primary antibody (1:1000 dilution) overnight at 4 °C, followed by a HRP-conjugated secondary antibody (1:1000 dilution in blocking buffer). Proteins of interest were visualized with ECL western blotting substrate according to manufacturer’s instructions (GE Healthcare, Uppsala, Sweden). The following antibodies were used: KEAP1 (10503–2-AP, Protein Tech, Chicago, IL), NRF2 (2178–1, Epitomics, Burlingame, CA), HMOX1 (ab13243, Abcam, Cambridge, England), NQO1 (11451, Protein Tech, Chicago, IL), GCLM (sc22754, Santa Cruz Biotechnology, Dallas, TX), BRG1 (G7, sc17796, Santa Cruz Biotechnology, Dallas TX), BRM (ab15597, Abcam, Cambridge, England) and β-ACTIN (A2066, Sigma, St. Louis, MO).

Song S., Nguyen V., Schrank T., Mulvaney K., Walter V., Wei D., Orvis T., Desai N., Zhang J., Hayes D.N., Zheng Y., Major M.B, & Weissman B.E. (2020). Loss of SWI/SNF chromatin remodeling alters NRF2 signaling in non-small cell lung carcinoma. Molecular cancer research : MCR, 18(12), 1777-1788.

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Western Blot Analysis of MetRS-521 Fragments

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Tetra-His (BSA-Free, Qiagen) and mTOR (human FRB Domain, Enzo Life Sciences) antibodies were used to detect the MetRS-521 N- and C-terminal fragments, respectively. Total protein analyzed using SDS-PAGE was transferred to Protran nitrocellulose (Whatman), washed with 20 mL of TBST buffer (100 mM Tris pH 7.5, 150 mM NaCl, and 0.1% Tween 20) for 5 min three times and blocked with 10% dry milk. Membranes containing identical samples were incubated with each antibody (1:1000 and 1:2000 dilutions, respectively) for 1 hour, and the membranes were incubated with a 1:1000 dilution of a secondary goat anti-mouse IgG conjugated to peroxidase (Calbiochem) and a 1:5000 dilution of a secondary goat anti-rabbit IgG conjugated to peroxidase (Calbiochem), respectively. The signals were visualized using the ECL Western blotting substrate (GE Healthcare) and imaged using film (Bio-Excell).

Thomas E.E., Pandey N., Knudsen S., Ball Z.T, & Silberg J.J. (2017). Programming post-translational control over the metabolic labeling of cellular proteins with a non-canonical amino acid. ACS synthetic biology, 6(8), 1572-1583.

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Western Blot Analysis of RhoGAP Proteins

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RAW 264.7 cells transfected with siRNA directed against one of the three RhoGAP candidates or with a non-targeting siRNA control were washed with PBS and lysed with cold RIPA buffer (Sigma-Aldrich) supplemented with protease- and phosphatase-inhibitor tablets (Roche). 40 μg of total protein were loaded per lane of a 12% SDS–polyacrylamide gel electrophoresis and separated by electrophoresis, before being transferred to methanol-activated polyvinylidene fluoride membranes. Membranes were blocked in Tris-buffered saline (TBS) supplemented with 0.1% Tween 20 and 5% bovine serum albumin for 30 min. This solution was also used to dilute the primary anti-RhoGAP and anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies, which were incubated for 2 h and 45 min, respectively. Primary antibodies against ARHGAP12, ARHGAP25 and SH3BP1 were used at 1:250, 1:1,000 and 1:1,000 dilutions, respectively. HRP-conjugated antibodies were used at a 1:10,000 dilution. Membranes exposed to ECL western blotting substrate (GE Life Sciences) were visualized using the Odyssey Fc (LI-COR) system. Brightness/contrast parameters were adjusted globally across the entire image using the LI-COR Image Studio software. Immunoblots were cropped for presentation; see Supplementary Fig. 6 for uncropped images.

Schlam D., Bagshaw R.D., Freeman S.A., Collins R.F., Pawson T., Fairn G.D, & Grinstein S. (2015). Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins. Nature Communications, 6, 8623.

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Western Blot Analysis of Protein Targets

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Cells or tissues were lysed in RIPA buffer (Tris–HCl 50 mM, pH 7.4, NaCl 150 mM, sodium deoxycholate 0.25%, NP-40 1%, EDTA 1 mM, PMSF 1 mM, Aprotinin 1 mg/ml, leupeptin 1 mg/ml, pepstain 1 mg/ml) and a total of 20 ug of protein was separated by SDS-PAGE electrophoresis and transferred to PVDF membranes (Amersham International, GE Healthcare). Membranes were incubated with blocking solution (5% milk powder in tris-buffered saline–Tween 20 (TBST) ) for 1 h, then with primary antibody (in blocking solution) overnight at 4°C. After several washes in TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature (RT) in blocking solution. Membranes were incubated with ECL western-blotting substrate (Amersham International, GE Healthcare) and imaged by in a ChemiDOC XRS system or ChemiDOC (Bio-Rad Laboratories).
The following antibodies were used in this study: anti-HA-tag antibody (MBL, M180–3) for CjCas9, β-tubulin mouse antibody (Proteintech, 66240-1-Ig), β-actin antibody (Sigma-Aldrich, A3854), UCP1 antibody (Abcam, ab10983), FGF21 (Abcam, ab171941).

Zhang X., Lv S., Luo Z., Hu Y., Peng X., Lv J., Zhao S., Feng J., Huang G., Wan Q.L., Liu J., Huang H., Luan B., Wang D., Zhao X., Lin Y., Zhou Q., Zhang Z.N, & Rong Z. (2021). MiniCAFE, a CRISPR/Cas9-based compact and potent transcriptional activator, elicits gene expression in vivo. Nucleic Acids Research, 49(7), 4171-4185.

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Western Blot Analysis of Islet Oxidative Stress

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Hypoxic WT and Nox2^−/− islets were harvested and whole cell extracts were generated, as described previously in detail [34 (link)]. Protein extracts were then separated through a 12.5% SDS polyacrylamide gel and transferred onto a PVDF membrane. The membrane was incubated in 5% dry milk in phosphate-buffered saline (PBS) (0.1% Tween20) for 1 h and exposed to anti-Nfr2, anti–HO–1, anti-SOD1 and SOD2, anti-insulin and anti-β-actin antibodies, which were diluted (1:500) in PBS (0.1% Tween20) containing 1% dry milk. After incubation of the membrane with a peroxidase-coupled secondary antibody (anti-rabbit 1:2000 or anti-mouse 1:2000) for 1 h, the protein expression was visualized by the incubation of the membrane with enhanced chemiluminescence (ECL) Western blotting substrate (GE Healthcare, Solingen, Germany) in a Chemocam device (Intas, Göttingen, Germany). The intensity of the measured signals was quantified using ImageJ software and normalized by the corresponding housekeeping protein.

Wrublewsky S., Glas J., Carlein C., Nalbach L., Hoffmann M.D., Pack M., Vilas-Boas E.A., Ribot N., Kappl R., Menger M.D., Laschke M.W., Ampofo E, & Roma L.P. (2022). The loss of pancreatic islet NADPH oxidase (NOX)2 improves islet transplantation. Redox Biology, 55, 102419.

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Immunoblotting Protein Expression Analysis

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Immunoblotting was performed as previously described (31 ). Protein lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF. The membrane was incubated in blocking buffer and then with a primary antibody overnight at 4°C. After washing, the membrane was incubated with an HRP-conjugated secondary antibody in blocking buffer. The primary antibodies included: BRG1 (A300-813A, Bethyl laboratories), CDH1 (610281, BD Transduction Laboratories), CDH3 (2130, Cell Signaling technology), CD44 (Dr. Larry Sherman, Oregon Health Sciences University), CK18 (DC-10, SCBT), RRAD (Dr. C. Ronald Kahn, Joslin Diabetes Center and Harvard Medical School), and β-ACTIN (A2066, Sigma). Proteins of interest were visualized with ECL Western blotting substrate (GE Healthcare) or a CCD camera imaging system (ChemiDoc™ XRS+, BIO-RAD).

Song S., Walter V., Karaca M., Li Y., Bartlett C.S., Smiraglia D.J., Serber D., Sproul C.D., Plass C., Zhang J., Hayes D.N., Zheng Y, & Weissman B.E. (2014). Gene Silencing Associated with SWI/SNF Complex Loss During NSCLC Development. Molecular cancer research : MCR, 12(4), 560-570.

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Western Blot Analysis of Fragmented IFP

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E. coli expressing the different fragmented IFP were grown as described
for fluorescent analysis, harvested by centrifugation, and resuspended
to identical optical densities. Sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) was carried out under reducing conditions
using NuPAGE 12% Bis-Tris Gels (Life Technologies) and MOPS SDS running
buffer and transferred to Protran nitrocellulose membrane (Whatman)
using a TE 22 mini Tank Transfer Unit (GE Healthcare). After washing
the nitrocellulose paper in TBST buffer (100 mM Tris, pH 7.5, 150
mM NaCl, 0.1% Tween-20) for 5 min and blocking for 1 h with 10% dry
milk in TBST, the membranes were incubated for 1 h with either GST
rabbit (Millipore) or hemagglutinin Ab-1 (NeoMarkers) polyclonal antibodies
at dilutions of 1:10000 in TBST. The nitrocellulose was then incubated
for 1 h in TBST with secondary antibody, goat anti-rabbit IgG conjugated
to peroxidase conjugate (Calbiochem), at a dilution of 1:10000. Signals
were detected using the ECL western blotting substrate (GE Healthcare)
according to the manufacturer’s protocol.

Pandey N., Nobles C.L., Zechiedrich L., Maresso A.W, & Silberg J.J. (2014). Combining Random Gene Fission and Rational Gene Fusion To Discover Near-Infrared Fluorescent Protein Fragments That Report on Protein–Protein Interactions. ACS Synthetic Biology, 4(5), 615-624.

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Western Blot Analysis of Nrf2 Pathway

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A549 or MCF7 cells treated with MSU38225 were lysed in RIPA buffer (5 M NaCl, 1 M Tris-Cl, pH 7.4, 0.5 M EDTA, 25 mM deoxycholic acid, 1% triton-X, 0.1% SDS) containing protease inhibitors (1 mM PMSF, 2 μg/ml aprotinin and 5 μg/ml leupeptin) added just prior to use. Protein concentrations were measured using the BCA assay (Sigma-Aldrich). 20 μg of protein were separated by 10% SDS-PAGE gels and transferred to nitrocellulose membranes. Nrf2 (Novus Biologicals, 1:1000), NQO1 (Abcam, 1:1000), HO-1 (Abcam, 1:1000), Keap1 (Cell Signaling Technology, 1:1000), NF-κB (Cell Signaling Technology, 1:1000), IκBα (Cell Signaling Technology, 1:1000) Cyclin G1 (Santa Cruz, 1:1000), β-TrCP (Cell Signaling Technology, 1:1000), STAT3 (Cell Signaling Technology, 1:1000), Ubiquitin (Cell Signaling Technology, 1:1000), Histone 3 (Cell Signaling Technology, 1:4000), GAPDH (Santa Cruz, 1:4000) and Vinculin (Cell Signaling Technology, 1:4000) primary antibodies were applied to detect the corresponding proteins. Secondary antibodies (anti-rabbit or anti-mouse linked to HRP, 1:1000) were purchased from Cell Signaling Technology. ECL Western blotting substrate (GE Healthcare Life Sciences, UK) was used to detect the signal. Images shown are representative of 3 independent experiments. Protein quantification was done using ImageJ.

Zhang D., Hou Z., Aldrich K.E., Lockwood L., Odom A.L, & Liby K.T. (2021). A novel Nrf2 pathway inhibitor sensitizes Keap1-mutant lung cancer cells to chemotherapy. Molecular cancer therapeutics, 20(9), 1692-1701.

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Liver Protein Extraction and Western Blot Analysis

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Nuclear and cytoplasmic extracts were generated from whole liver using
previously described buffers with protease and phosphatase inhibitors^{53 (link)}. Samples were run on SDS-PAGE
4–20% Tris-Glycine gradient gels, electrophoretically transferred to
nitrocellulose membrane and probed with antibodies. Signals were detected by ECL
Western blotting substrate (GE Healthcare). The membrane was stripped and
reprobed with anti-actin antibody to verify and normalize protein loading using
densitometry. Quantification was performed using ImageJ software.

Schaub J.R., Huppert K.A., Kurial S.N., Hsu B.Y., Cast A.E., Donnelly B., Karns R.A., Chen F., Rezvani M., Luu H.Y., Mattis A.N., Rougemont A.L., Rosenthal P., Huppert S.S, & Willenbring H. (2018). De novo formation of the biliary system by TGFβ-mediated hepatocyte transdifferentiation. Nature, 557(7704), 247-251.

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