Ecl chemiluminescent substrate

Manufactured by Biosharp

Sourced in China

ECL chemiluminescent substrate is a reagent used for the detection of proteins in Western blotting applications. It emits light upon reaction with the enzyme horseradish peroxidase (HRP), which is commonly used to label target proteins. The emitted light can be captured and quantified using a suitable imaging system, allowing for the visualization and analysis of the target proteins.

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Lab products found in correlation

Pvdf membrane, by Merck Group (2 mentions) Chemidoc mp chemiluminescence gel imaging system, by Bio-Rad (1 mentions) Runx2, by ABclonal (1 mentions) Collagen 1 col 1, by Abcam (1 mentions) Protease inhibitor cocktail, by Beyotime (1 mentions) Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions) Gapdh, by ABclonal (1 mentions) Anti collagen 3, by Abcam (1 mentions) Bca protein assay reagent, by Beyotime (1 mentions) Anti collagen 1, by Cell Signaling Technology (1 mentions) Bs 20412r, by Bioss Antibodies (1 mentions) Anti p smad2, by Cell Signaling Technology (1 mentions) Anti p smad3, by Cell Signaling Technology (1 mentions) Anti α sma, by Cell Signaling Technology (1 mentions) Sa00001 2, by Proteintech (1 mentions) Anti β actin antibody, by Abcam (1 mentions) Ripa lysis buffer, by Biosharp (1 mentions) Bca protein assay kit, by Beyotime (1 mentions) Chemidoc xr, by Bio-Rad (1 mentions) Bca protein concentration assay kit, by Biosharp (1 mentions)

4 protocols using ecl chemiluminescent substrate

Protein Isolation and Western Blot Analysis of Bone Tissues

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Bone tissues
were placed in a mortar, and liquid nitrogen was added, allowing the
callus to be ground to fine particles with a pestle. Subsequently,
the tissue was transferred to a 1.5 mL EP tube for protein isolation.
Total protein was extracted from cells and tissues by RIPA (Beyotime,
P0013B, Shanghai, China) with 1% protease inhibitor cocktail for general
use, 100× (Beyotime, P1005, Shanghai, China). Protein (20 μg)
was separated on a denatured sodium dodecyl sulfate-polyacrylamide
gel and transferred to a polyvinylidene fluoride (PVDF) membrane.
The PVDF membrane was blocked with defat mike (5% dissolved in TBS-T)
for 2 h and then incubated with rabbit polyclonal ALP (1:1000, Abcam),
Runx2 (1:1000, Abclonal), OCN (1:500, Abcam), collagen I (Col1) (1:3000,
Abcam), GAPDH (1:1000, Abclonal), SSRP1 (1; 1000, Proteintech), and
β-catenin (1:1000, Abclonal) for 16 h at 4 °C. After being
washed three times, the membranes were followed by incubation with
horseradish peroxidase-coupled goat antirabbit IgG H&L for 1 h
at 37 °C. The blotting membrane was treated with an ECL chemiluminescent
substrate (Biosharp, BL520A, Shanghai, China) and visualized using
a ChemiDoc MP chemiluminescence gel imaging system (Bio-Rad,1708280,
California, USA).

Yu C., Chen L., Zhou W., Hu L., Xie X., Lin Z., Panayi A.C., Zhan X., Tao R., Mi B, & Liu G. (2022). Injectable Bacteria-Sensitive Hydrogel Promotes Repair of Infected Fractures via Sustained Release of miRNA Antagonist. ACS Applied Materials & Interfaces, 14(30), 34427-34442.

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Quantitative Western Blot Analysis of Cardiac Fibrosis

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Western blot analysis was performed as described by us (Wang et al., 2019b (link)). Briefly, proteins was extracted from CFs with RIPA lysis buffer (Biosharp, Hefie, China) and quantified with a BCA protein assay reagent (Beyotime, Haimen, China). Equal of protein lysates was loaded into per lane for SDS-PAGE detection before transferred to PVDF membranes (Millipore, Billerica, MA, USA). Next, all membranes were blocked with 5% nonfat milk solution in TBST buffer for 1 h at room temperature and then incubated with primary antibodies on a shaker at 4 °C overnight. The primary anti-bodies: Anti-Collagen I (Cell Signaling Technology, 91144, dilution: 1:1,000), Anti-Collagen III (Abcam, ab7778, dilution: 1:7,000), Anti-α-SMA (Cell Signaling Technology, 19245, dilution: 1:1,000), Anti-p-Smad2 (Cell Signaling Technology, 18338, dilution: 1:1,000), Anti-p-Smad3 (Cell Signaling Technology, 9520, dilution: 1:1,000), Anti-TGF-β2 (Bioss, bs-20412R, dilution: 1:1,000) and Anti-β-actin Antibody (Abcam, ab8227, dilution: 1:1,000), were used. The next day, membranes were incubated with secondary antibody (Proteintech, SA00001-2, dilution: 1:5,000) for 1 h at room temperature. And then the membranes were detected with ECL chemiluminescent substrate (Biosharp, Hefie, China). β-actin acted as the internal standard.

Liu W., Wang Y., Qiu Z., Zhao R., Liu Z., Chen W., Ge J, & Shi B. (2020). CircHIPK3 regulates cardiac fibroblast proliferation, migration and phenotypic switching through the miR-152-3p/TGF-β2 axis under hypoxia. PeerJ, 8, e9796.

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Western Blot Protein Quantification

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Total protein was extracted from tissues or cells using RIPA lysis buffer (Servicbio.Inc., Wuhan, China) with 1% protease inhibitor Cocktail (MCE). After that, the protein concentration was adjusted to the same level using a BCA protein assay kit (Beyotime Biotechnology). After the electrophoresis and transmembrane, PVDF membranes (Millipore, Bedford, USA) were first incubated in 5% BSA (Servicbio.Inc.) for 2 h, then transferred to primary antibodies overnight at 4 °C after being rinsed with PBS. Next, the membranes were incubated in corresponding secondary antibodies for 1 h. Finally, the protein bands were detected using ChemiDoc XRS+ (Bio‑Rad Laboratories, CA, USA) with ECL chemiluminescent substrate (Biosharp, Wuhan, China). Information of the antibodies were shown in Supplementary Table 3.

Shou Y., Yue C., Wang Q., Liu J., Xu J., Miao Q., Liu D., Yang H., Liu Y, & Zhang X. (2023). circPTPN12 promotes the progression and sunitinib resistance of renal cancer via hnRNPM/IL-6/STAT3 pathway. Cell Death & Disease, 14(3), 232.

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Western Blot Analysis of BPTC Proteins

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The BPTCs treated with glucose with or without the addition of 100 nmol/L rapamycin for 24 h were collected for western blotting analysis. The BPTCs were washed three times with PBS, and 1 mL of cell RIPA solution was added to obtain total protein. The protein concentration was detected by the BCA protein concentration assay kit (Biosharp, Guangzhou, China). Western blot analysis was performed in brief as follows: 20 µg of protein/well was separated on 6–15% separating gel, 2–10% concentrating gel (Servicebio, Wuhan, China) and transferred to PVDF membrane (Absin, Shanghai, China). The membranes were sequentially closed with 5% skimmed milk generated in Tris-buffer, followed by incubation using primary antibodies, overnight at 4 °C. The membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody for 1 h at room temperature. Subsequently, the membranes were incubated with the specific ultrasensitive ECL chemiluminescent substrate (Biosharp, Guangzhou, China) and visualization of the proteins was achieved with the ChemiDOC MP (Bio-Rad). The mean values of protein in 1 mg/mL treatment were set to 1.00. Grayscale analysis was performed using ImageJ Software 1.8.0. The complete details of primary antibodies are listed in Table 2.

Shi L., Kang K., Wang Z., Wang J., Xiao J., Peng Q., Hu R., Zhou J., Zhang X., Yue Z., Zou H., Xue B, & Wang L. (2023). Glucose Regulates Glucose Transport and Metabolism via mTOR Signaling Pathway in Bovine Placental Trophoblast Cells. Animals : an Open Access Journal from MDPI, 14(1), 40.

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