Thermo ecl substrate

Manufactured by Bio-Rad

Thermo ECL Substrate is a chemiluminescent reagent used for the detection of proteins in Western blot analysis. It generates a luminescent signal when combined with a horseradish peroxidase (HRP)-labeled detection system.

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Lab products found in correlation

β actin, by Abcam (2 mentions) β actin antibody, by Abcam (1 mentions) Anti β catenin mab, by Abcam (1 mentions) Ab32503, by Abcam (1 mentions) Ab254360, by Abcam (1 mentions) Ab183218, by Abcam (1 mentions) Ab62352, by Abcam (1 mentions) Ab205718, by Abcam (1 mentions) Ab9485, by Abcam (1 mentions) Ab32124, by Abcam (1 mentions) Ab263899, by Abcam (1 mentions) P foxo1, by Abcam (1 mentions) Pvdf membrane, by Merck Group (1 mentions) P akt, by Abcam (1 mentions) T akt, by Abcam (1 mentions)

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ECL substrate (255 citations)

5 protocols using thermo ecl substrate

Western Blot Analysis of FOXN3, β-Catenin, and TCF4

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Protein was extracted from C666-1 cells using radio-immunoprecipitation assay (RIPA) buffer containing PMSF. Samples were placed on ice for 20 min at 4°C and then centrifuged at 12 000 rpm for 20 min. The supernatant (200 μL) was collected and placed in an EP tube. Total protein concentration was determined using the BCA Protein Assay Kit PC0020-500 (Solaibao Technology Co. LTD, Beijing, China). Protein were separated by a SDS-polyacrylamide gel according to previously reported methods.^{28 (link),
29 (link)} After blocking, blots were washed with TBS containing 0.5% Tween-20 (Beyotime Institute of Biotechnology, Jiangsu, China) and incubated overnight at 4°C with the anti-FOXN3 mAb (1:1500, Abcam, Beijing, China), anti-β-Catenin mAb (1:1500, Abcam, Beijing, China) or anti-TCF4 mAb (1:1000, Abcam, Beijing, China). After several washes with Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated in a 1:5000 dilution of an anti-mouse HRP-conjugated secondary antibody for 2 h. Subsequently, the signal was detected by chemiluminescence using the Thermo ECL Substrate (Bio-Rad). The same membrane was incubated with a β-actin antibody (1:1000, Abcam, Beijing, China), which served as a loading control. The results were collected via the Versa DocTM imaging system (Peiqing Technology Co. LTD, Shanghai, China) and analyzed with Image J software.

Lin Z., Chen M., Wan Y., Lei L, & Ruan H. (2020). miR-574-5p Targets FOXN3 to Regulate the Invasion of Nasopharyngeal Carcinoma Cells via Wnt/β-Catenin Pathway. Technology in Cancer Research & Treatment, 19, 1533033820971659.

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Western Blot Analysis of Protein Expression

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Radio immunoprecipitation assay (RIPA) buffer containing the PMSF was used to dissolve brain tissues. After centrifugation, the supernatant was used for protein concentration detection. The same amount of protein was applied for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE). The proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, and blocked with 5% skimmed milk (3 h). The membrane was washed with PBST, and incubated with related primary antibodies (1:800) overnight at 4°C. After washing with PBS twice, the membrane was incubated with second antibody (1:2000) for 3 h. Chemiluminescence with Thermo ECL Substrate (Bio‐Rad) was used to incubate the membrane, and ImageJ software was used to analyze the band gray. The antibodies used in this study were listed as follows: anti‐Bax antibody (ab32503; Abcam), anti‐nuclear factor erythroid 2‐related factor 2 (Nrf2) antibody (ab62352; Abcam), anti‐B‐Cell CLL/Lymphoma 2 (Bcl‐2) antibody (ab32124; Abcam), antitumor necrosis factor (TNF)‐α antibody (ab183218; Abcam), anti‐NLRP3 antibody (ab263899; Abcam), anti‐IL‐1β antibody (ab254360; Abcam), anti‐Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody (ab9485; Abcam), and goat anti‐Rabbit IgG (ab205718; Abcam).

Liu Y., Rao K., Li Z, & Huang C. (2023). Improvement of neurological recovery in the insomnia rats by Warming Yang Strategy through targeting SIRT4 by inhibiting inflammation and apoptosis. Immunity, Inflammation and Disease, 11(8), e964.

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Western Blot Analysis of Hep3B Cells

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Protein samples were extracted from Hep3B cells with the RIPA buffer, including PMSF, on ice for 20 min at 4°C. The supernatant solution (200 μL) was collected in an EP tube after centrifuging at 12 000 rpm for 20 min. The BCA Protein Assay Kit PC0020-500 (Solaibao Technology Co. Ltd, Beijing, China) was used to detect the protein concentration. Proteins were separated using SDS-polyacrylamide gel step according to the previous description [20 (link), 21 (link)] and the separated protein was blocked. The blocking protein was washed with TBS buffer, including 0.5% Tween-20 (Zolgene Biotechnology Co., Ltd, Fuzhou, China), and incubated overnight at 4°C with anti-FOXC2 MMP-2, MMP-9, and β-actin (1 : 1500, Abcam, Beijing, China). Primary antibodies were washed with Tris-buffered saline containing 0.1% Tween-20 after incubation. Samples were incubated with an antimouse HRP-conjugated secondary antibody (1 : 5000 dilution) for 2 h. The signal was detected using chemiluminescence with Thermo ECL Substrate (Bio-Rad). Membranes incubated with β-actin (1 : 1000, Abcam, Beijing, China) were used as loading controls. The Versa DocTM imaging system (Peiqing Technology Co. Ltd, Shanghai, China) was utilised to collect the results, which were analyzed with Image J software.

Weng Z., Peng J., Wu W., Zhang C., Zhao J, & Gao H. (2021). Downregulation of PART1 Inhibits Proliferation and Differentiation of Hep3B Cells by Targeting hsa-miR-3529-3p/FOXC2 Axis. Journal of Oncology, 2021, 7792223.

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Western Blot Protein Analysis

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Cells were lysed with radio immunoprecipitation assay (RIPA) lysate with 1% phenylmethanesulfonyl fluoride (PMSF). Bicinchoninic acid (BCA) commercial kit was used to measure protein content. 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. After transferring to the PVDF membrane, which was blocked using 10% skimmed milk (2 h). The PVDF membranes were cultured with primary antibodies overnight at 4°C. The PVDF membranes were cultured with secondary antibody for 4 h. The proteins were measured with chemiluminescence with Thermo ECL Substrate (Bio-Rad). The protein bands were analyzed using ImageJ software.

Wu W., Chen X., Hu Q., Wang X., Zhu J, & Li Q. (2022). Improvement of Myocardial Cell Injury by miR-199a-3p/mTOR Axis through Regulating Cell Apoptosis and Autophagy. Journal of Immunology Research, 2022, 1642301.

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Protein Expression Profiling in Cells

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The protein samples were extracted from the cells with RIPA buffer including the PMSF. The supernatant solution was collected after centrifuging at 8 000 rpm for 10 min. Same amounts of proteins were separated with SDS-PAGE. Then, the proteins were transferred to a PVDF membrane (Sigma, USA). After blocking with TBST, the membrane was incubated with anti-GLUC2, T-AKT, p-AKT, p-FOXO1, T-FOXO1, Pdx1, and β-actin (1 : 1500, Abcam, Beijing, China) overnight at 4°C. After washing twice, the membrane was incubated with secondary antibody (antimouse HRP-conjugated antibody, 1 : 2000) for 1 h. Finally, the protein was detected by chemiluminescence with Thermo ECL Substrate (Bio-Rad) and analyzed using the ImageJ software.

Zhang Y., Li L., Zhang Y., Yan S, & Huang L. (2022). Improvement of Lipotoxicity-Induced Islet β Cellular Insulin Secretion Disorder by Osteocalcin. Journal of Diabetes Research, 2022, 3025538.

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