After being irradiated, the cells were incubated for 24 h and then lysed with protease inhibitor. Aliquots of cell lysates were loaded at adequate amounts onto gels and then subjected to electrophoresis. Samples was then transferred to a polyvinylidene fluoride membrane (PVDF, Merck Millipore, Burlington, MA, USA) and further incubated with primary antibodies to phosphorylated ATM (pATM, GeneTex, Irvine, CA, USA), poly (ADP-ribose) polymerase-1 (PARP1, Cell Signaling Technology, Danvers, MA, USA), γ-H2AX (GeneTex), and β-tubulin (Merck Millipore, Burlington, MA, USA). Bound antibodies were detected using appropriate peroxidase-coupled secondary antibodies followed by enhanced chemiluminescence (ECL, Boehringer Mannheim, Mannheim, Germany). Images were obtained after washing out the secondary antibody. The fluorescence images were visually scanned for luminescence and quantitatively analyzed using ImageJ software.
Enhanced chemi luminescence (ecl)
Manufactured by Roche
Sourced in Germany, Switzerland, China, United States
ECL is a laboratory equipment product that is used for electrochemiluminescence (ECL) detection. ECL is a highly sensitive detection technique that generates light through an electrochemical reaction, enabling the quantification of a wide range of analytes, including proteins, nucleic acids, and small molecules. The core function of ECL is to provide a reliable and efficient method for sensitive and quantitative analysis in various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (8 mentions)
X ray film, by Thermo Fisher Scientific (5 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Protein assay, by Bio-Rad (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Supersignal west femto substrate, by Thermo Fisher Scientific (3 mentions)
Gradient gel, by Bio-Rad (3 mentions)
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Bradford assay kit, by Merck Group (2 mentions)
Pefabloc sc, by Merck Group (2 mentions)
Protease inhibitor tablet, by Roche (2 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (2 mentions)
Peroxidase conjugated secondary antibody, by Abcam (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
β tubulin, by Merck Group (1 mentions)
Poly adp ribose polymerase 1 parp1, by Cell Signaling Technology (1 mentions)
γ h2ax, by GeneTex (1 mentions)
Phospho h2ax ser139, by Merck Group (1 mentions)
39 protocols using enhanced chemi luminescence (ecl)
1
Quantifying DNA Damage Response Proteins
2
Western Blot Analysis of Signaling Proteins
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The proteins (50 μg of protein/aliquot) in aliquots of cell lysates were separated by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE; 8–15% polyacrylamide), transferred to polyvinylidene difluoride (PVDF) membranes, and immunoblotted with various antibodies. Bound antibodies were detected using appropriate peroxidase-coupled secondary antibodies followed by enhanced chemiluminescence (ECL, Boehringer Mannheim, Mannheim, Germany). Antibodies to phospho-Akt and phospho-mTOR were obtained from Cell Signaling, Inc. (Burlingame, CA, USA), phospho-Akt (Ser473 and Thr308) and phospho-mTOR (Ser2448), phosphor-p70 S6 kinase (Thr389) and caspase-3 from Cell Signaling Technology (Danvers, MA, USA), S6 and beta-actin from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and phospho-H2AX (Ser139) from Millipore Corporation (Billerica, MA, USA). Beta-actin was used as the loading control.
3
Western Blot Analysis of Ovarian Cells
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Each treatment within each culture was replicated for at least 3 times. The granulosa cells (1 × 106 cells/well) were cultured in 6-well plate. Cells were lysed using a lysis buffer (1% Triton X-100, 150 mM, NaCl, 1 mM EGTA, 1% NP-40, 1 mM NaF, 1 mM Na3VO4, 2 mM phenylmethyl- sulfonylfuoride (PMSF), 1 mg/ml aprotinin and leupeptin in PBS) and centrifuged at 12,000 rpm for 25 min at 4 °C. For ovarian tissue protein extraction, each ovarian tissue was homogenized and extracted in lysis buffer. The cell lysates or tissue lysates were purified and quantified using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). A total of 50-μg protein sample was separated using SDS-PAGE, transferred onto a polyvinylidene difluoride (PVDF) membrane, and immunoblotted with various antibodies. Bound antibodies were detected using the appropriate peroxidase-coupled secondary antibodies and an enhanced chemiluminescence detection system (ECL, Boehringer Mannheim, Indianapolis, IN, USA).
4
Western Blot Analysis of Cell Signaling
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After cells were treated with various radiation doses and drugs, total protein was collected at different times. Protein was extracted using Mammalian Protein Extraction Reagent (M-PER; Pierce, Rockford, Ill.). An equal amount of protein from each cell line was loaded per lane and separated on a 10 % sodium dodecyl sulfate (SDS)–Tris glycine polyacrylamide gel electrophoresis (PAGE) gel. Gels were transferred onto nitrocellulose membranes (Novex, San Diego, Calif.) and blocked overnight by incubating with 1 × Tris-buffered saline containing 0.1 % Tween and 5 % nonfat dry milk. Membranes were probed with antibodies against phospho-Akt (p-Akt; Cell Signaling, Beverly, Mass.), phospho-mTOR (p-mTOR; Novus International, St. Louis, Mo.), phospho-eIF4E (p-4EBP), phospho–p70 ribosomal protein S6 kinases (p-p70S6K; Cell Signaling), phospho–eukaryotic translation initiation factor 4E (p-eIF4E; Cell Signaling), VEGF-C (Zymed Laboratories, South San Francisco, Calif.), Akt, mTOR, 4EBP, eIF4E, and actin (all from Chemicon International, Temecula, Calif.). Bound antibodies were then detected using the appropriate peroxidase-coupled secondary antibodies, followed by incubation with an enhanced chemiluminescence detection system (ECL; Boehringer Mannheim, Mannheim, Germany).
5
Western Blot Analysis of p53 and p21 in Mouse Granulosa Cells
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Mouse granulosa cells (1 × 106 cells/well) were cultured in 6-well plates. Cells were lysed using a lysis buffer (1% Triton X-100, 150 mM, NaCl, 1 mM EGTA, 1% NP-40, 1 mM NaF, 1 mM Na3VO4, 2 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mg/ml aprotinin and leupeptin in PBS) and centrifuged at 13 200g (12 000rpm; Kubota Model 3500) for 25 min at 4°C. The cell lysates were quantified using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). A total of 50-μg protein per sample was separated using SDS-PAGE, transferred onto a polyvinylidene difluoride (PVDF) membrane and immunoblotted with anti-p53 antibody (sc-393031, 1/1000), anti-p-p53 (S392) (sc-7997, 1/1000) or anti-p21 antibody (sc-817, 1/1000). Bound antibodies were detected using appropriate peroxidase-coupled secondary antibodies, an enhanced chemiluminescence detection system (ECL, Boehringer Mannheim, Indianapolis, IN, USA) and a Digital imaging system (Bio Pioneer Tech Co., New Taipei City, Taiwan). Furthermore, the membranes incubated with anti-p53 antibody were stripped in the following buffer (37.5 mM Tris, pH 6.8, 2% SDS and 1% β-mercaptoethanol) at 56°C for 20 min. Stripped membranes were washed three times using PBS with Tween (10 mM Tris, pH 7.5,150 mM NaCl, 0.05% Tween 20), followed by immunoblotting with anti-alpha-tubulin (α-t) antibody (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA).
6
Western Blot Analysis of p-AKT/AKT in HUVECs
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After treatment with the different combinations of VEGF and IL-10, the HUVECs were submitted for Western blotting to determine the expression of p-AKT/AKT. The HUVECs were lysed using a lysis buffer (Thermo Fisher Scientific) and centrifuged at 12,000 rpm for 25 min at 4 °C. The cell lysates were quantified using a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). A total of 50 μg of protein per sample was collected using SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes, and immune-blotted with antibodies for p-AKT and AKT, respectively. Antibody bindings were detected using the appropriate peroxidase-coupled secondary antibodies and an enhanced chemiluminescence detection system (ECL, Boehringer Mannheim, Indianapolis, IN, USA). Photographs were obtained using a digital imaging system (Bio Pioneer Tech Co., New Taipei City, Taiwan).
7
BCL10 Expression Analysis in PDAC Cells
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The shBCL10 untreated and treated PDAC cells were plated on cell culture slide (SPL Life Sciences) overnight. After washing twice with PBS, the cells xed with 3.7% paraformaldehyde at 4°C for 8 min, permeabilized with 0.1% TritonX-100 in PBS for 10 min, and blocked with 3% BSA at room temperature for 1 h. Then, cells were incubated with the anti-BCL10 primary antibody ( [19, 21] . The speci c reactive bands on membranes were probed using appropriate secondary IgG antibodies conjugated to horse radish peroxidase. The immune complexes were visualized using an enhanced chemiluminescence detection system (ECL, Boehringer Mannheim, Mannheim) and quanti cation was performed using the Image Quant software (GE Healthcare). All experiments were repeated at least three times.
8
Protein Isolation and Western Blot Analysis
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Total protein or nuclear protein was isolated with a commercial lysis buffer (Pierce, Thermo Scientific) following standard instructions. Protein was frozen at -20°C before use. Protein concentrations were quantified by BCA assays (Pierce BCA Protein Assay Kit, Thermo Scientific), and samples were adjusted to the same concentration with lysis buffer. For each sample, the same quantity of protein (10 to 30 μg) was subjected to 10% or 12% SDS-PAGE, then proteins separated on the gel were transferred to a nitrocellulose membrane and blocked with 5% defatted milk/TBST for 1 h at room temperature. The membrane was incubated with primary antibodies at 4°C overnight. The primary antibodies were diluted in 5% defatted milk/TBST with specific dilutions as follows: Nrf2 (1:1000), PPARGC1a (1:1000), complex I (1:2000), complex II (1:4000), complex III (1:4000), complex IV (1:1000), complex V (1:4000), catalase (1:1000), GCLc (1:1000), NQO-1 (1:2000), GST(1:500), β-actin (1:10000), and α-tubulin (1:10000). After washing membranes with TBST three times, membranes were incubated with horseradish peroxidase-conjugated antibody for 1 h at room temperature. Western blots were developed using ECL (Roche Manheim, Germany) and quantitative analysis based on optical density was performed with Quantity One software.
9
Protein Extraction and Detection Protocol
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Total proteins were extracted in 1% NP-40, 0.5% sodium-deoxycholate, 0.1% SDS supplemented with protease inhibitor cocktail from Roche Diagnostics (Mannheim, Germany). The proteins linked to the chromatin were extracted as described in Hervouet et al.38 (link) Protein concentration was determined using Bradford assay (Bio-Rad, Hercules, CA, USA). Protein extracts were separated by SDS-PAGE, transferred onto PVDF membrane (Millipore, St. Quentin-Yvelines, France) and revealed with ECL (Roche Diagnostics). HRP-conjugated secondary antibodies were from Bio-Rad.
10
Western Blot Analysis of Smad Signaling
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Three pellets per donor and group were homogenized mechanically with a Mixer Mill MM 400 (Retsch, Haan, Germany) and resuspended in PhosphoSafeTM Extraction Reagent and 1 mM Pefabloc®SC (both Sigma-Aldrich). Cell lysates were centrifuged at 13000 rpm and 4 °C for 20 min and concentration was determined using Bradford assay kit (Sigma-Aldrich). Lysates were separated by SDS-PAGE gel electrophoresis and transferred to a nitrocellulose membrane (GE Healthcare). After blocking for 1 h at 20 °C with 5% skim milk in 0.1% TBST (25 mM Tris/HCl pH 7.4; 145 mM NaCl, 2.7 mM KCl, 0.1% Tween 20) the membrane was incubated over night at 4 °C with primary antibody. The β-Actin (1:10000, GeneTex, GTX26276/18985), pSmad2 (1:250, Cell Signalling, #3108/7), Smad2/3 (1:250, Cell Signalling, #8685/3), pSmad 1/5/9 (1:250, Cell Signalling, #9511/18), Smad1 (1:500, Abcam, ab33902/GR1487581) and Smad5 (1:1000, Abcam, ab40771/YK05017CS) antibody was diluted in 5% skim milk in 0.1% TBST. Next, the blots were washed with 1% TBST and incubated with anti-mouse (Jackson Immuno Research, 115-035-071/29454) or anti-rabbit (Jackson Immuno Research, 111-035-046/27362) secondary antibodies in 5% skim milk in 0.1% TBST for an hour at 20 °C. After washing with 0.1% TBST, the blots were developed with ECL (Roche).
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