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Enhanced chemiluminescence ecl substrate

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Enhanced chemiluminescence (ECL) substrate is a reagent used for detecting and quantifying proteins in Western blot analysis. It generates a luminescent signal when exposed to the enzyme-labeled secondary antibody, allowing for the visualization and analysis of target proteins.

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12 protocols using enhanced chemiluminescence ecl substrate

1

Protein Expression Analysis of Tumor Samples

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Cells and mechanically dissociated tumor samples were lysed in RIPA buffer (Thermo) with protease-phosphatase inhibitor cocktail (Sigma) and cleared by centrifugation. Proteins were quantified by Bradford reagent assay or BCA assay. WB was performed using our laboratory optimized protocol (17 (link)). Membranes were blotted with anti-CD206 (CST#91992), anti-CD163 (CST#93498), anti-PD-L1 (CST#13684), anti-Snail (CST#3895), anti-Slug (CST#9585), anti-E-cadherin (CST#14472) and anti-β-actin (CST#4970) antibodies. Immunoreactive bands were developed using horse radish peroxidase (HRP)-conjugated secondary antibodies (CST#7074, CST#7076) and Enhanced Chemiluminescence (ECL) substrate (Thermo).
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2

Quantifying Myocardial Protein Expression

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Protein expression levels of ACE, ACE2, AT1R, and MasR in cardiac tissue were determined using western blotting as previously described [24 (link)]. In brief, frozen cardiac tissues were thawed, ground, and homogenized in the lysis buffer that contained protease inhibitors, surfactants, as well as phosphatase inhibitors. Protein samples were loaded in 8% or 10% gels for sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The resolved proteins were electrically transferred to the nitrocellulose membranes and detected using specific antibodies, including anti-ACE (SantaCruz Biotechnology, Inc., CA, USA; cat no. sc-23,908; lot no. C1319), anti-ACE2 (Abcam, UK; cat no. ab108252; lot no. GR145000–28), anti-AT1R (SantaCruz Biotechnology, Inc., CA, USA; cat no. sc515884; lot no. J0319), and anti-MasR (SantaCruz Biotechnology, Inc., CA, USA; cat no. sc-390,453; lot no. A1419), to quantify myocardial protein levels. The primary antibody-antigen complexes and the horseradish peroxidase (HRP)-conjugated secondary antibodies were incubated and then visualized using an enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific Inc., USA) and ECL hyperfilm (GE Healthcare Pvt. Ltd., UK). Blots were inspected by ImageJ software (NIH, USA). Results were normalized with the beta-actin (Sigma-Aldrich, USA; cat no. A5441) as the internal standard.
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3

Western Blot Analysis of Protein Expression

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RIPA lysate was used for extracting the total proteins from the tissue samples and HCC cells, which were quantified with the help of a BCA kit. About 30 μg of total proteins were then separated by way of 100 g/L SDS-PAGE gel electrophoresis. The segregated proteins were moved to a PVDF membrane and blocked using 50 g/L skim milk at room temperature for 2 h. The membranes were incubated with the primary antibodies specific for HMGA2 (CST, 8179, 1:1000 dilution), CD68 (Abcam, ab125212, 1:2000 dilution), CD163 (Abcam, ab182422, 1:1000 dilution), CD204 (Abcam, ab123946, 1:2500 dilution), Histone H3 (CST, 4499, 1:2000 dilution), or GAPDH (CST, 5174, 1:1000 dilution). Further incubation of the blots was conducted utilizing goat anti-rabbit IgG and goat anti-mouse IgG, followed by detection to visualize the bands with the help of the enhanced chemiluminescence (ECL) substrate (Thermo, Waltham, MA, USA).
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4

Wnt-3a Signaling Pathway Assay

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Dimethyl sulfoxide (DMSO) and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) were purchased from Sigma‐Aldrich (St. Louis, MO, USA). Recombinant human Wnt‐3a protein was purchased from R&D systems (Minneapolis, MN, USA). β‐catenin, α‐tubulin antibodies, horseradish peroxidase‐conjugated mouse IgGκ binding protein (m‐IgGκ BP‐HRP), and CruzFluor 488‐conjugated mouse IgGκ binding protein (m‐IgGκ BP‐CFL 488) were obtained from Santa Cruz Biotechnology (CA, USA). The bicinchoninic acid (BCA) assay kit and enhanced chemiluminescence (ECL) substrate were obtained from Thermo scientific (Rockford, IL, USA). Alexa Fluor 555 streptavidin was obtained from Invitrogen (CA, USA), and 4,6′‐diamidino‐2‐phenylindole (DAPI) was purchased from Vector Laboratories (Burlingame, CA, USA).
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5

Chemical Compounds Analysis Protocol

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MEHP, rapamycin, and bafilomycin A1 were purchased from Sigma-Aldrich (St. Louis, MO). Wortmannin and staurosporine were purchased from Cell Signaling Technology Inc. (Danvers, MA). Chemicals were dissolved in DMSO (Sigma Aldrich, St Louis MO), to a final concentration of 0.1% DMSO. Dulbecco’s modified Eagle’s medium (DMEM), DMEM/F12, StemPro medium, and fetal bovine serum (FBS) were purchased from Thermo-Fisher Scientific Inc. (Waltham, MA). Collagenase Type IV with trypsin activity, hyaluronidase, trypsin, and PBS were obtained from Sigma-Aldrich. The enhanced chemiluminescence (ECL) substrate was purchased from Thermo-Fisher Scientific Inc. The polyvinylidene difluoride (PVDF) membranes were obtained from Bio-Rad.
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6

Immunoblotting for HA-tagged Proteins

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Cell lysates of 1pG cells and 8pG cells were electrophoresed in 10% reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a nitrocellulose membrane. The blots were blocked in 5% milk in phosphate-buffered saline with Tween 20 (PBST) and incubated with mouse anti-HA monoclonal IgG antibody (1 μg/ml, clone MMS-101R, Biolegend, San Diego, CA, U.S.A). HRP-conjugated goat anti-mouse IgG F(ab’)2 antibodies (1 μg/ml) and enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific, Waltham, MA, U.S.A.) were used to detect protein signals.
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7

Western Blot Analysis of Protein Biomarkers

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The cell lysates were prepared using radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific, Inc.) and protease inhibitor cocktail (GenDEPOT, Barker, TX, USA). Cell lysates were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. Protein-transferred membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline Tween 20 (TBS-T) for 1 h at room temperature and incubated with blocking buffer-diluted primary antibodies, including E-cadherin (Abcam; ab76055), iNOS (Abcam; ab178945), c-Met (Abcam; ab51067), VEGFA (Abcam; ab46154), Ki-67 (Abcam; ab16667) and ABCB1 (Abcam; ab170904) antibody (1:1000) at 4°C overnight. After the membranes were washed and incubated with secondary antibodies, immunoreactive protein expression signals were detected using the enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific, Inc.) and visualized using Luminograph III (ATTO Corporation, Tokyo, Japan). The expression of anti-β-actin (Sigma-Aldrich Co.) was used as the loading control.
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8

NF-κB Pathway Activation Analysis

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Dulbecco's modified Eagle's medium was purchased from Gibco (Grand Island, NY, USA). Fetal bovine serum was from Sijiqing (Hangzhou, Zhejiang, China). Penicillin, streptomycin, and nuclear and cytoplasmic extraction kit were from Shenggong (Shanghai, China). Abs to phospho-IκB-α, IκB-α, NF-κB-p65, β-actin, and JSH-23 were all from Santa Cruz (Santa Cruz, CA, USA). PDTC, MG-132, BAY 11-7082, BCA protein assay kit, nitric-oxide synthase assay kit, and abs to histone H3 were from Beyotime (Nantong, Jiangsu, China). Abs specific to mouse TLR4 was from eBioscience (San Diego, CA, USA). LPS, o-phenylenediamine, fluoresceinamine (FLA), and 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) were from Sigma-Aldrich (St. Louis, MO, USA). Enhanced chemiluminescence (ECL) substrate was from Thermo Scientific.
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9

Western Blot Analysis of Cell Signaling

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After the indicated treatments, cells were collected and lysed in cell lysis buffer (Cell Signaling Technology). Protein concentrations were determined using a Bradford protein assay kit (Thermo Fisher Scientific, Waltham, USA). Proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, USA). Anti-p-JNK (1:2000), anti-JNK (1:2000), anti-ERK1/2 (1:2000), anti-p-ERK1/2 (1:2000), anti-p-p38 (1:2000), anti-p38 (1:2000), anti-Bax (1:2000), anti-caspase3 (1:1000) and anti-β-actin (1:8000) antibodies were used as primary antibodies to incubated with the membrane. After incubation with an appropriate HRP-conjugated secondary antibody (1:4000), protein bands were visualized using an enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific), detected using a Bio-Rad imaging system (Bio-Rad, Hercules, USA), and quantified with ImageJ software (NIH, Bethesda USA).
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10

Western Blot Analysis of M. synoviae

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Purified recombinant proteins, M. synoviae whole cells, or M. synoviae fractions were solubilized in sample buffer (50 mM Tris-HCl pH 6.8, 10% glycerol, 0.1%BPB, 1% 2-β-mercaptoethanol, and 2% SDS) and subjected to 12% SDS-PAGE. For western blot analysis, the protein was electrotransferred onto a PVDF membrane (Immun-Blot® PVDF, Bio-Rad) at 70 V for 90 min. The membranes were incubated in blocking buffer (5% skim milk in PBST) overnight at 4 °C. After washing with PBST, the membranes were primary incubated with the primary antibody for 2 h at 37 °C. Then, they were incubated with goat anti-rabbit or goat anti-mouse IgG-HRP (H + L) antibody (1∶5000 dilution) for 2 h at 37°C. The specific bands were detected using an enhanced chemiluminescence (ECL) substrate (Thermo Fisher). Equal volumes of Reagent A (luminol) and Reagent B (an enhancer) were gently added to the membrane surface after liquid mixing, and images were captured using a chemiluminescence detection system (Tianneng, Shanghai, China).
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