Ecl developer

Manufactured by Thermo Fisher Scientific

Sourced in United States

The ECL developer is a laboratory equipment designed to develop and detect chemiluminescent signals in Western blot analysis. It utilizes enhanced chemiluminescence (ECL) technology to facilitate the visualization of protein bands on a membrane.

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Lab products found in correlation

Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Odyssey gel imaging system, by Thermo Fisher Scientific (2 mentions) Ab59479, by Abcam (1 mentions) Ab79559, by Abcam (1 mentions) Ab133682, by Abcam (1 mentions) Ab8226, by Abcam (1 mentions) Proteojet mammalian cell lysis reagent, by Thermo Fisher Scientific (1 mentions) P0015, by Beyotime (1 mentions) Sds page electrophoresis solution, by Beyotime (1 mentions) Trizol lysis buffer, by Thermo Fisher Scientific (1 mentions) Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions) Tb green premix ex taq 2, by Takara Bio (1 mentions) Sds page gel preparation kit, by Solarbio (1 mentions) Bca protein concentration assay kit, by Beyotime (1 mentions) D8340, by Solarbio (1 mentions) Skimmed milk powder, by Solarbio (1 mentions) 0.22 μm pvdf membrane, by Merck Group (1 mentions) Gel imaging system, by Bio-Rad (1 mentions) Phosstop, by Roche (1 mentions) Polyvinylidene difluoride pvdf membrane, by Thermo Fisher Scientific (1 mentions)

7 protocols using ecl developer

Quantitative Protein Expression Analysis

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The total protein of the myocardium was extracted, and its concentration was determined by the BCA method (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific). Tissue protein was separated by 15% SDS-PAGE and then wetted to PVDF membrane (Millipore), 5% BSA closed at room temperature for 2 h, plus anti-primary (1 : 1000 dilution) and incubated overnight at 4°C in a refrigerator, and finally washed and incubated with goat anti-rabbit IgG (Proteintech Group) at room temperature for 2 h. Reactive bands of the PVDF membrane were equipped with ECL developer (Thermo Fisher Scientific) and placed in a BIO-RAD gel imager to image, and the Image Lab software calculated its gray value. The process was repeated three times, and the results were presented as the ratio of the gray value of the target protein band to the GAPDH band. The first antibodies used were monoclonal rabbit anti-Smad2 antibody (Cell Signaling Technology), monoclonal rabbit anti-p-Smad2 antibody (Cell Signaling Technology), monoclonal rabbit anti-TGF-β1 antibody (Cell Signaling Technology), and HRP-conjugated monoclonal mouse anti-GAPDH antibody (Shanghai KangChen Bio-tech Co. Ltd., China) (loading control).
Reactive bands were equipped with ECL developer (Thermo Fisher Scientific) which immediately drops on the PVDF membrane, placed in BIO-RAD gel imager imaging and image Lab software to calculate its gray value.

Wu T., Xie Y., Huang J., Li P., Wang X., Yan Y., Xia T., Li L., Zhu F., Li H, & Wu R. (2017). The Optimal Intervention Time of Bone Marrow Mesenchymal Stem Cells in Ameliorating Cardiac Fibrosis Induced by Viral Myocarditis: A Randomized Controlled Trial in Mice. Stem Cells International, 2017, 3258035.

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Protein Extraction and Western Blot Analysis

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ProteoJET™ Mammalian Cell Lysis Reagent (Thermo Scientific, Waltham, MA) was added to extract the cell protein of each group, and the protein was quantified by the BCA method. Proteins (50 μg) were separated using 10% SDS-PAGE and transferred to PVDF membranes. The PVDF membranes were blocked with 5% non-fat dry milk in TBST for 1 hour, then probed with the specific primary antibodies at 4 ℃ for 24 h, and incubated with the second antibody at room temperature for 1 h. Finally, ECL developer (Thermo Scientific) was added and exposure imaging was performed by Odyssey gel imaging system. Mouse polyclonal antibody against NUPR1L was prepared by Genex Health Co., Ltd., and the specific antibodies to PFDN1 (ab151708), CD63 (ab59479), CD81 (ab79559), Integrin α6 (ab97760), Integrin β4 (ab133682) and β-actin (ab8226) were purchased from Abcam (Cambridge, UK).

Gao Q., Lei F., Zeng Q., Gao Z., Niu P., J.u.n.n.a.n., N.i.n.g., Li J, & Zhang J. (2020). Functional Passenger-Strand miRNAs in Exosomes Derived from Human Colon Cancer Cells and Their Heterogeneous Paracrine Effects. International Journal of Biological Sciences, 16(6), 1044-1058.

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Comprehensive Protein Analysis Protocol

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The main reagents used in this study included TRIzol lysis buffer (Invitrogen, USA); reverse transcription kit: PrimeScript RT reagent kit with GDNA Eraser (RR 047A, Takara, Japan); qPCR kit: Tb Green Premix EX TAQ II (RR 820A, Takara); animal whole protein extraction kit (C510003, Sangon Biotech, China); BCA protein concentration assay kit (P0010, Beyotime Biotech, China); 5× loading buffer (P0015, Beyotime Biotech); SDS-PAGE gel preparation kit (P1200, Solar BioBeijing, China); SDS-PAGE electrophoresis solution (P00148, Beyotime Biotech); 10× EMT (D1060, Solar BioBeijing); methanol (CB/T693-1993, Shanghai Guangnuo Chemical Technology Co., Ltd., China); skimmed milk powder (D8340, Solar BioBeijing); 10×TBST buffer (powder) (T1087, Solar BioBeijing); nitrocellulose membrane (NC membrane) (37412133, PALL, USA); and ECL developer (34095, Thermo Fisher, USA).

Wang J., Hu Y., Xu Y., Long Q., Gu C., Tang C., Wang R, & Yong S. (2024). Phospholipase D regulates ferroptosis signal transduction in mouse spleen hypoxia response. Brazilian Journal of Medical and Biological Research, 57, e13218.

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Western Blot Analysis of Protein Signaling

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Total protein was extracted from homogenized NRC and myocardial tissue samples, and phosphatase inhibitor (PhosSTOP, Roche) was added on the basis of the manufacturer's instructions. The protein concentration was measured by BCA assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific). The proteins were separated by 12% SDS-PAGE and wetly transferred to 0.22 μm PVDF membrane (Millipore). After 2h of continued blocking by 5% skim milk dissolved in TBS, the membranes were, respectively, incubated in monoclonal antibodies, including pAkt (1 : 1000, Ser473, #4060, Rabbit mAb, CST), Akt (1 : 1000, #4691, Rabbit mAb, CST), survivin (1 : 1000, #2808, Rabbit mAb, CST), Cleaved Caspse-3 (1 : 1000, #9661, Rabbit mAb, CST), Caspase-3 (1 : 1000, #9665, Rabbit mAb, CST), and β-actin (1 : 1000, ab8227, Rabbit mAb), at 4°C overnight. The membranes were then washed in TBST (0.1% Tween 20 in TBS) and incubated with goat anti-rabbit IgG (CST) for 2 h at room temperature (RT). The ECL developer (Thermo Fisher Scientific) was used, and the images were analyzed by the Bio-Rad Gel Imaging System, while the gray value was measured with Image Lab software.

Li P., Yan Y., Shi Y., Cheng B., Zhan Y., Wang Q., Ye Q., Weng Y., Wu T, & Wu R. (2019). Nicotinic Agonist Inhibits Cardiomyocyte Apoptosis in CVB3-Induced Myocarditis via α3β4-nAChR/PI3K/Akt-Dependent Survivin Upregulation. Oxidative Medicine and Cellular Longevity, 2019, 9496419.

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Western Blot Analysis of MLPH in RAW 264.7 Cells

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Raw 264.7 cells treated with coal dust (200 μg/mL) and 1.25(OH)₂VD₃ (1.2 × 10^–10 mg/mL) were harvested, and cell lysis was obtained in RIPA buffer (Beyotime, Shanghai, China). Supernatants were collected, followed by centrifuging at a speed of 10,000 rpm for 10 min, and the protein concentration was determined using a BCA protein concentration determination kit (Beyotime, Shanghai, China). Next, 30 μg of the protein sample was loaded on an SDS-PAGE gel and electrophoresed. The electrophoresed proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific, Waltham, MA, USA). After blocking, MLPH antibody or GAPDH (Cell Signaling Technology, Danvers, MA, USA) was added. Chemiluminescence was then used to detect the proteins with an ECL developer (Thermo Fisher Scientific, Waltham, MA, USA). Finally, the protein expression levels were quantitated using ImageJ software.

Mu M., Li B., Zou Y., Wang W., Cao H., Zhang Y., Sun Q., Chen H., Ge D., Tao H., Hu D., Yuan L., Tao X, & Wang J. (2022). Coal dust exposure triggers heterogeneity of transcriptional profiles in mouse pneumoconiosis and Vitamin D remedies. Particle and Fibre Toxicology, 19, 7.

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Breast Cancer Cell Lines Analysis

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The main experimental materials and reagents were as follows: human BC cell lines MCF-7, BT-20, MDA-MB-231, and MCF-10A and human normal breast epithelial cells Hs 278Bst (purchased from ATCC), qRT-PCR and reverse transcription kits (TransGen Biotech, Beijing, China), CCK-8 kits (Promega, USA), Transwell kit (Beijing Xinshengyuan Biomedical Technology Co., Ltd), PBS, fetal bovine serum (FBS) (Gibco, Rockville, MD, USA), Trizol reagent (Beijing Baiaolaibo Technology Co., Ltd), Dual-Lucy Assay kit (Beijing Baiaolaibo Technology Co., Ltd), p-PI3K, p-AKT, N-cadherin, E-cadherin, vimentin, and β-Actin antibodies (Cell Signaling Technology, Boston, MA, USA), goat anti-rabbit IgG secondary antibody (Wuhan Boster Biology Co., Ltd), RIPA, BCA protein kit (Thermo Fisher Scientific, Waltham, MA, USA), ECL developer (Thermo Fisher Scientific, Waltham, MA, USA), PCR instrument (ABI, USA). All primers were designed and compounded by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

Sheng X.Y., Wang C.H., Wang C.F, & Xu H.Y. (2020). Long-Chain Non-Coding SOX21-AS1 Promotes Proliferation and Migration of Breast Cancer Cells Through the PI3K/AKT Signaling Pathway. Cancer Management and Research, 12, 11005-11014.

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Protein Extraction and Western Blot Analysis

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Total exosomic and cellular protein was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology, Beijing, China). The protein concentrations of cell and exosome lysates were determined using BCA protein assay kit (Beyotime Institute of Biotechnology, Beijing, China). For Western blot analyses, the cellular and exosomic proteins (10 µg/well) were separated by electrophoresis in 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membrane (Amersham, Buckinghamshire, UK). The membrane was blocked with 5% skimmed milk in TBS-T (10 mM TrisCl, pH 8.0, 150 mM NaCl, 0.5% Tween 20) at 4°C overnight, rinsed 3 times (10 min/time) with TBS-T, followed by 3 h incubation at room temperature with the specific primary antibodies, followed by 1 h incubation with the second antibody. ECL developer (Thermo Scientific) was added and exposure imaging was performed by Odyssey gel imaging system. The specific antibodies to E-cadherin (ab1416), N-cadherin (ab76011), Vimentin (ab92547), Snail (ab216347), CD81 (ab109201), HSP70 (ab181606), TSG101 (ab125011), Calnexin (ab133615), were purchased from Abcam (Cambridge, UK). β-actin antibody (#4970) was acquired from Cell Signaling Technology (Danvers, MA, USA).

Chen C., Ma Z, & Jiang H. (2021). EMT Participates in the Regulation of Exosomes Secretion and Function in Esophageal Cancer Cells. Technology in Cancer Research & Treatment, 20, 15330338211033077.

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