Enhanced chemiluminescence ecl

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Enhanced chemiluminescence (ECL) is a laboratory technique used for the detection and quantification of proteins in Western blot analysis. It is a sensitive method that utilizes the principle of chemiluminescence to generate a light signal that can be detected and measured. The ECL reaction involves the oxidation of a chemiluminescent substrate, which results in the emission of light that can be captured and quantified using a suitable imaging system.

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Lab products found in correlation

Alexa fluor 488 annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (1 mentions) Erp72, by Cell Signaling Technology (1 mentions) Bca protein estimation kit, by Merck Group (1 mentions) Alamar blue, by Merck Group (1 mentions) β actin, by Cell Signaling Technology (1 mentions) P erk, by Cell Signaling Technology (1 mentions) Insulin, by Merck Group (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Lipopolysaccharides lps from escherichia coli 0111 b4, by Merck Group (1 mentions) Dimethyl sulfoxide (dmso), by Santa Cruz Biotechnology (1 mentions) L glutamine, by Thermo Fisher Scientific (1 mentions) Ficoll paque plus, by GE Healthcare (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Anti β tubulin, by Merck Group (1 mentions) 0.2 μm nitrocellulose membrane, by Bio-Rad (1 mentions) Chemidoc xrs imaging system, by Bio-Rad (1 mentions) Ab32518, by Abcam (1 mentions) Ab32135, by Abcam (1 mentions)

5 protocols using enhanced chemiluminescence ecl

Evaluating Gambogic Acid's Anticancer Potential

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GA was purchased from Nanjing Zelang Medical Technology Co., Ltd. (Jiangsu, China), and dissolved in dimethyl sulfoxide (DMSO) (Sigma, St. Louis, MO, USA) to make a stock solution before use. For treatment of cells, it was diluted in culture medium at the appropriate concentrations, and the final concentration of DMSO was <0.01% (v/v). Cisplatin (Lot no. H20030675; Nanjing Pharmaceutical Factory Co., Ltd., Jiangsu, China), and insulin, propidium iodide (PI), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and alamarBlue were from Sigma. Alexa Fluor 488 Annexin V/Dead Cell Apoptosis kit was from Invitrogen Life Technologies (Carlsbad, CA, USA). Antibodies against caspase-3, -7 and -9, p18, p16, p27, p21, cyclin-D1, -D3 and -E2, CDK6, 4 and 2, E2F-1, pRb, Bip, PERK, ERP72, β-actin, and HRP-conjugated antibodies (anti-rabbit or mouse immunoglobulin G) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA).
BCA protein estimation kit was from Sigma. Nitrocellulose (NC) blotting membrane was from Pall Corporation (DF Mexico, Mexico). Enhanced chemiluminescence (ECL) was from Bio-Rad (Hercules, CA, USA).

ZHU J., CHEN M., CHEN N., MA A., ZHU C., ZHAO R., JIANG M., ZHOU J., YE L., FU H, & ZHANG X. (2015). Glycyrrhetinic acid induces G1-phase cell cycle arrest in human non-small cell lung cancer cells through endoplasmic reticulum stress pathway. International Journal of Oncology, 46(3), 981-988.

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Immune Cell Isolation and Stimulation

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RPMI 1640 medium, fetal bovine serum (FBS), L-glutamine, and penicillin/streptomycin were from Gibco by Life Technologies, UK. Ficoll-Paque Plus was from GE Healthcare, Sweden. Phosphate-buffered saline (PBS) was from HyClone, Logan, Utah. Dimethyl sulfoxide (DMSO) was from Santa Cruz Biotechnology, USA. PP2 (4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d] pyrimidine) was from Sigma Aldrich, USA. Lipopolysaccharides (LPS) from Escherichia coli 0111:B4 were from Sigma Aldrich, USA. BD vacutainer heparinized tubes were from BD Biosciences, UK. Enhanced chemiluminescence (ECL) was obtained from Bio-Rad, USA.

Irtegun S., Pektanc G., Akkurt Z.M., Bozkurt M., Turkcu F.M, & Kalkanli-Tas S. (2016). Pharmacological Inactivation of Src Family Kinases Inhibits LPS-Induced TNF-α Production in PBMC of Patients with Behçet's Disease. Mediators of Inflammation, 2016, 5414369.

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Quantifying IDO1 Protein Degradation

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Transfected B16 cells were incubated with the protein synthesis inhibitor cycloheximide 40 μg/ml for the time-course analysis of IDO1 protein degradation. Whole-cell lysates (1 x 10⁵ cells/sample) were run on SDS/PAGE and electro-blotted onto 0.2 μm nitrocellulose membranes (Bio-Rad, CA, USA). Membranes were probed with a monoclonal antibody specific for mouse IDO1 (Millipore), in combination with an appropriate horseradish peroxidase-conjugated antibody (Millipore), followed by enhanced chemiluminescence (ECL) (Bio-Rad). Anti-β-tubulin (Sigma-Aldrich) was used as a normalizer. Densitometric analysis of specific signals was performed by ChemiDoc XRS+Imaging System (Bio-Rad), within a linear range of blot exposures, selecting the two lowest exposure times required for detecting signals.

Orecchini E., Belladonna M., Pallotta M., Volpi C., Zizi L., Panfili E., Gargaro M., Fallarino F., Rossini S., Suvieri C., Macchiarulo A., Bicciato S., Mondanelli G, & Orabona C. (2023). The signaling function of IDO1 incites the malignant progression of mouse B16 melanoma. Oncoimmunology, 12(1), 2170095.

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Anti-inflammatory Effects of DHA Lipid Mediators

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FO (EPA C20:5 n-3: 60 mg/g, DHA, C22:6, n-3: 460 mg/g, DPA, C22:5 n-3 + n-6, 105 mg/g) was acquired from KD Nutra (Branttvag, Norway). A mixture of lipid mediators (LM) was obtained from DHA through enzymatic reaction using soybean lipoxygenase, which is composed of 17S-monohydroxy DHA, resolvin D5, and protectin DX at ratio of 3:47:50. Palmitic acid (PA) was purchased from Fluka (Seelze, Germany). Human liver cancer cell line HepG2 was purchased from Korea Cell Line Bank (Seoul, Republic of Korea). A bicinchoninic acid (BCA) assay kit was purchased from Sango Company (San Diego, CA, USA). Antibodies against IKKB (ab32135), p-IKKB (ab194528), IKB (ab32518), p-IKB (ab133462), NF-kB (p65) (ab16502), p-NF-kB (p-p65) (ab76302), and GAPDH (ab181602) were purchased from Abcam (Cambridge, MA, USA). Enhanced chemiluminescence (ECL) was purchased from Bio-Rad Laboratories, Inc. (Tewksbury, MA, USA). The assay kits for triacylglycerol (TG), total cholesterol (TC), high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol were purchased from BioVision (Milpitas, CA, USA). Mouse/human IL-6 and TNF-α ELISA kits were purchased from Abcam (Cambridge, MA, USA).

Su Y., Choi H.S., Choi J.H., Kim H.S., Lee G.Y., Cho H.W., Choi H., Jang Y.S, & Seo J.W. (2023). Effects of Fish Oil, Lipid Mediators, Derived from Docosahexaenoic Acid, and Their Co-Treatment against Lipid Metabolism Dysfunction and Inflammation in HFD Mice and HepG2 Cells. Nutrients, 15(2), 427.

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Western Blot Analysis of Protein Lysates

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Tissue was lysed in RIPA buffer containing protease and phosphatase inhibitors (Halt protease and phosphatase inhibitor, Thermo Scientific, Waltham, MA, USA). Protein (30-40µg) was resolved by polyacrylamide gel (8%) electrophoresis, then transferred to PDVF membranes, and incubated in blocking solution (5% bovine serum albumin TBS-Tween 0.05% in PBS) for 1h.
The membranes were incubated with primary antibodies overnight at 4C. On the following day, the membranes were incubated with secondary antibody for 1h at room temperature (RT) After several washes, blots were developed with Enhanced chemiluminescence ECL (BioRad, Hercules CA, USA). Bands were visualized using an imager developer (IMAGEQUANT LASc 4000, GE Healthcare Little Chalfont, UK). Images were quantified with ImageJ blots toolkit software (National Institutes of Health, Baltimore, MD, USA).

Sánchez-Sarasúa S., Meseguer-Beltrán M., García-Díaz C., Beltrán-Bretones M.T., ElMlili N, & Sánchez-Pérez A.M. (2022). IRS1 expression in hippocampus is age-dependent and is required for mature spine maintenance and neuritogenesis. Molecular and cellular neurosciences, 118.

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