Ecl western blot detection kit

Manufactured by GE Healthcare

Sourced in United States, United Kingdom

The ECL Western blot detection kit is a laboratory equipment used for the detection and quantification of proteins in biological samples. It utilizes enhanced chemiluminescence (ECL) technology to visualize and analyze protein expression levels.

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Lab products found in correlation

Ripa lysis buffer, by Beyotime (4 mentions) β actin, by Abcam (3 mentions) β actin, by Merck Group (2 mentions) Cleaved caspase 3, by Abcam (2 mentions) Bcl2 associated x (bax), by Abcam (2 mentions) Cyclin d1, by Abcam (2 mentions) Gapdh, by Merck Group (2 mentions) Ab1791, by Abcam (2 mentions) Ab8898, by Abcam (2 mentions) α tubulin a01410 40, by GenScript (2 mentions) S tag, by Fortis Life Sciences (2 mentions) Goat anti rabbit igg hrp, by Jackson ImmunoResearch (1 mentions) Anti rabbit or anti mouse igg, by Cell Signaling Technology (1 mentions) Polyvinylidene difluoride membrane, by Merck Group (1 mentions) Imagequant las 4000 mini instrument, by GE Healthcare (1 mentions) Las 4000, by Cytiva (1 mentions) Phosphatase inhibitor, by Beyotime (1 mentions) Horseradish peroxidase hrp labeled secondary antibody, by Merck Group (1 mentions) Polyvinylidene difluoride pvdf membrane, by Merck Group (1 mentions) Bicinchoninic acid bca kit, by Beyotime (1 mentions)

Spelling variants of this product

ECL kit (450 citations) ECL detection kit (236 citations)

23 protocols using ecl western blot detection kit

Western Blot Detection of CD200 Proteins

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After transfer onto PVDF membranes, the blots were blocked with 5% milk-TBST for 1 hour at room temperature, and then probed with primary antibodies overnight at 4°C. The following primary antibodies were used in Western blotting experiments: rabbit anti-hCD200v+c serum (1:2000 dilution), rabbit anti-CD200 c-tail serum (1:500 dilution), or rabbit-hCD200R1 serum (1:2000 dilution). Regardless of the primary antibody used, following primary antibody incubation and washings in TBS-T, all blots were probed with goat anti-rabbit IgG-HRP (Jackson) at a 1:10,000 dilution for 45 minutes as secondary antibody. After thorough washing, blots were developed using an ECL Western blot detection kit (GE Healthcare Bio-Sciences).

Wong K.K., Zhu F., Khatri I., Huo Q., Spaner D.E, & Gorczynski R.M. (2016). Characterization of CD200 Ectodomain Shedding. PLoS ONE, 11(4), e0152073.

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Western Blot Analysis of DNA Damage Signaling

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Equal amounts of proteins were fractionated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membranes were blocked and primary antibodies were added, followed by secondary antibodies. Signals were detected using an ImageQuant LAS 4000 Mini instrument (GE Healthcare, Wauwatosa, WI, USA).
HRP (horseradish peroxidase)-linked antibodies specific for ATR, phospho-ATR (Ser428), phosphor-Chk1 (Ser345), ATM, phospho-Chk2 (Thr68), phospho-H2AX (Ser139), and anti-mouse or anti-rabbit IgG were obtained from Cell Signaling Technology (Beverly, MA, USA). Antibodies specific for Chk1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), pATM from GeneTex (Irvine, CA, USA), Chk2 from Medical and Biological Laboratories (Nagoya, Japan), and β-actin from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were used according to the manufacturers’ recommendations.
Protein bands were detected by the ECL Western Blot Detection Kit (GE Healthcare Life Sciences, Piscataway, NJ, USA).
Molecular weight markers were used and all gel images with the markers are shown in the Supplementary Figure S1.

Takeuchi M., Tanikawa M., Nagasaka K., Oda K., Kawata Y., Oki S., Agapiti C., Sone K., Miyagawa Y., Hiraike H., Wada-Hiraike O., Kuramoto H., Ayabe T., Osuga Y, & Fujii T. (2019). Anti-Tumor Effect of Inhibition of DNA Damage Response Proteins, ATM and ATR, in Endometrial Cancer Cells. Cancers, 11(12), 1913.

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Protein Expression Analysis of Compound 7g in Colorectal Cancer

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Protein levels were analyzed by western blotting. Human colorectal cancer cell lines SW620 and HT29 treated with compound 7g for 48 h were collected and lysed in the lysis buffer containing RIPA (Radioimmunoprecipitation, Beyotime, Shanghai, China), a protease inhibitor and a phosphatase inhibitor (Roche, Mannheim, Germany). The concentration of proteins was measured by a BCA (Bicinchoninic acid) kit (Beyotime, P0010, Shanghai, China). Cell lysates (50 μg) were separated by 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore Corporation, MA, USA). The membranes were blocked in TBST containing 5% BSA (Beyotime, ST023, Shanghai, China) for 2 h at room temperature and then incubated with primary antibodies (diluted by product specification, CST, USA) overnight at 4 °C. After that, the membranes were washed five times with TBST and then incubated with horseradish peroxidase (HRP)-labeled secondary antibodies (diluted 1:10000, Sigma, USA) for 1 h at room temperature. Subsequently, membranes were washed five times with TBST and then visualized with the ECL western blot detection kit (GE Healthcare, RPN3244, USA) on a Tanon 5200 Imaging System (Tanon Science & Technology Co., Ltd, Shanghai, China).

He L.J., Yang D.L., Li S.Q., Zhang Y.J., Tang Y., Lei J., Frett B., Lin H.K., Li H.Y., Chen Z.Z, & Xu Z.G. (2018). Facile construction of fused benzimidazole-isoquinolinones that induce cell-cycle arrest and apoptosis in colorectal cancer cells. Bioorganic & medicinal chemistry, 26(14), 3899-3908.

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Quantification of RelB Protein Expression

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RelB protein expression in BMDCs was examined by Western blot. Briefly, extracted proteins from cell lysates were obtained using lysis buffer (Thermo Fisher Scientific, Massachusetts, USA) at 4° and were separated by 12% SDS–PAGE and then transferred to nitrocellulose membrane (Thermo Fisher Scientific). The membrane was blocked by 5% non‐fat milk in Tris‐buffered saline for 1 hr and then incubated with rabbit anti‐mouse RelB or rabbit anti‐mouse β‐actin polyclonal antibodies (Santa Cruz Biotech) for 1 hr at room temperature. This was followed by incubation with horseradish peroxidase‐goat anti‐rabbit IgG antibody (Cell Signaling, Danvers, MA). Protein bands were developed using an ECL Western Blot detection kit (GE Healthcare, Chalfont St Giles, UK). RelB was revealed as a protein band of 68 000 MW. Quantification was measured by densitometry and normalized to β‐actin level.

Wu H., Lo Y., Chan A., Law K.S, & Mok M.Y. (2016). Rel B‐modified dendritic cells possess tolerogenic phenotype and functions on lupus splenic lymphocytes in vitro. Immunology, 149(1), 48-61.

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Western Blot Analysis of HarA Protein

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Proteins were separated by SDS-PAGE and then electroblotted onto PVDF membranes (SequiBlot^TM, Bio-Rad Laboratories, Inc., Hercules, CA, USA). After blocking with 3% (w/v) skim milk, the blots were incubated with rabbit antiserum (diluted to 0.01%) directed against purified HarA and cell-free extracts of strain C-4A at 4 °C for 15 h, and horseradish peroxidase-conjugated secondary antibodies at room temperature for 80 min.
The immunoreactive proteins were detected with an ECL western blot detection kit (GE Healthcare). An uncropped image of the blot is shown in Supplementary Figure 31.

Nagakubo T., Kumano T., Ohta T., Hashimoto Y, & Kobayashi M. (2019). Copper amine oxidases catalyze the oxidative deamination and hydrolysis of cyclic imines. Nature Communications, 10, 413.

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Protein Extraction and Western Blot Analysis

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Total proteins were extracted and purified by using the commercial RIPA lysis buffer purchased from Beyotime Biotechnology (Shanghai, China). Next, a Bicinchoninic Acid Kit (Sigma, USA) was employed to determine total protein concentrations. The Western blot analysis was conducted according to the procedures provided by previous work.^{16 (link)} The primary Abs against Nrf2 (1:1000, Abcam, UK), β-actin (1:2000, Abcam, USA), TLR2 (1:1000, Abcam, UK), TLR4 (1:1000, Abcam, UK), TLR5 (1:1000, Abcam, UK), TLR9 (1:1500, Abcam, United Kingdom (UK)), Cyclin D1 (1:1000, Abcam, UK), CDK2 (1:1000, Abcam, UK), cleaved Caspase-3 (1:1500, Abcam, UK) and Bax (1:1500, Abcam, UK). The HRP-conjugated secondary Ab (1:2000) was also purchased from Abcam (UK). Finally, an ECL Western blot detection kit (GE Healthcare Bio-Sciences, USA) was used to visualize the protein bands, which were next quantified by detecting the grey values through using Image J software.

Tuoheti A., Gu X., Cheng X, & Zhang H. (2020). Silencing Nrf2 attenuates chronic suppurative otitis media by inhibiting pro-inflammatory cytokine secretion through up-regulating TLR4. Innate Immunity, 27(1), 70-80.

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Quantitative Analysis of CED-9 Protein

Cited 1 time

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A total of 300 synchronized wild-type N2 worms at day 1 and day 4 adult stages supplemented with DMSO or DIM for 24 h were harvested in 10 μL of protease inhibitor cocktail per experiment and lysed at 100 °C for 10 min. Each sample (18 μL) was loaded onto a 4–12% SDS-PAGE gel (Thermo Fisher Scientific, Waltham, MA, USA) and transferred to nitrocellulose membrane (PROTRAN BA83, Whatman, Sigma-Aldrich, St. Louis, MO, USA), and the procedure was followed as previously described [22 (link)]. Primary antibodies used were rabbit anti-CED-9 (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) and mouse anti-α-tubulin (1:1000; Sigma-Aldrich, St. Louis, MO, USA). The secondary antibodies used were horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) and HRP-conjugated donkey anti-mouse IgG (1:1000; Jackson ImmunoResearch, West Grove, PA, USA). Blots were visualized with an ECL western blot detection kit (Amersham, GE Healthcare Life Sciences, Pittsburgh, PA, USA), detected using the LAS-3000 image analyzer, and quantified using ImageJ software.

Lee M., Youn E., Kang K, & Shim Y.H. (2022). 3,3′-Diindolylmethane Supplementation Maintains Oocyte Quality by Reducing Oxidative Stress and CEP-1/p53-Mediated Regulation of Germ Cells in a Reproductively Aged Caenorhabditis elegans Model. Antioxidants, 11(5), 950.

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Transient Transfection and Immunoblotting in HEK293T Cells

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Adherent HEK293T cells were maintained under standard conditions and transfected (see Supplemental Table S1 for plasmids) with linear polyethylenimine (PEI; Polysciences, Eppelheim, Germany) in a 1:3 DNA:PEI ratio. Cells were collected after 48 h, lysed in 100 mM Tris-HCl, pH 8, 150 mM NaCl, and 0.1% NP-40 supplemented with 1/1000 of the EDTA-free protease inhibitor cocktail Set III (Calbiochem, Darmstadt, Germany), and centrifuged for 10 min, 15,000 × g at 4°C. SDS–PAGE and immunoblotting onto nitrocellulose membranes (EMD Millipore, Darmstadt, Germany) were performed using standard protocols. Proteins were detected using different primary antibodies (Supplemental Table S2) and visualized with horseradish peroxidase–labeled secondary antibodies, followed by chemiluminescence (ECL Western blot detection kit; GE Healthcare, Velizy-Villacoublay, France).

Tort O., Tanco S., Rocha C., Bièche I., Seixas C., Bosc C., Andrieux A., Moutin M.J., Avilés F.X., Lorenzo J, & Janke C. (2014). The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids. Molecular Biology of the Cell, 25(19), 3017-3027.

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RIPA Lysis and Western Blot Analysis

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The radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) was used to extract the total protein from cancer tissues and cells. Western Blot was conducted to determine the expression levels of proteins involved in this study according to the previous study [18 (link)]. The primary antibodies against NLRP3 (1:1000), β-actin (1:1000) and ASC (1:1500) were purchased from Abcam (UK). The protein bands were visualized by using the electro-chemi-luminescence (ECL) Western Blot detection kit (GE Healthcare Bio-science, USA) in keeping with the manufacturer’s instruction and quantified by Image J software.

Ren N., Jiang T., Wang C., Xie S., Xing Y., Piao D., Zhang T, & Zhu Y. (2020). LncRNA ADAMTS9-AS2 inhibits gastric cancer (GC) development and sensitizes chemoresistant GC cells to cisplatin by regulating miR-223-3p/NLRP3 axis. Aging (Albany NY), 12(11), 11025-11041.

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Western Blot Analysis of Recombinant Proteins

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The purified recombinant proteins (4 µg/lane) were subjected to SDS-PAGE using 10% gels and then transferred to polyvinylidene difluoride membranes. Non-specific binding was blocked for 1 h with 2% bovine serum albumin (BSA) in PBS (pH 7.4) containing 0.1% Tween 20 at 25 °C. The membranes were incubated overnight in a solution containing anti-His antibody (Santa Cruz Biotechnology, sc-803) or anti-c-myc antibody (Santa Cruz Biotechnology, sc-40). The bands were visualized with ECL western blot detection kit (GE Healthcare)^{35 (link)}.

Seo W.Y., Kim J.H., Baek D.S., Kim S.J., Kang S., Yang W.S., Song J.A., Lee M.S., Kim S, & Kim Y.S. (2017). Production of recombinant human procollagen type I C-terminal propeptide and establishment of a sandwich ELISA for quantification. Scientific Reports, 7, 15946.

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