Ecl procedure

Manufactured by Merck Group

Sourced in United States

The ECL procedure is a laboratory technique used for the detection and quantification of specific proteins in a sample. It relies on a chemiluminescent reaction to generate a measurable signal that is proportional to the amount of the target protein present. The core function of the ECL procedure is to provide a sensitive and reliable method for protein analysis in various research and diagnostic applications.

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5 protocols using ecl procedure

Protein Extraction and TLR5 Expression Analysis

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Total protein was extracted from cultivated RASFs using RIPA lysis buffer (Beyotime Institute of Biotechnology), and protein concentration was determined using the BCA method. TLR5 protein expression was measured using western blotting, which was performed according to standard procedures (25 (link)). A total of 20 µg was protein per lane lane and separated using 8-10% SDS-PAGE. PVDF membranes (EMD Millipore) were blocked with 3% BSA (Sigma-Aldrich; Merck KGaA) at 25˚C for 1 h. β-actin on the same membrane was used as the loading control. The antibodies were purchased from Abcam and were as follows: anti-TLR5 (1:2,000; cat. no. ab168382), anti-β-actin (1:10,000; cat. no. ab227387;), horse-radish peroxidase (HRP)-labelled anti-Rabbit IgG (1:50,000; cat. no. ab205718). Primary antibody incubation was at 4˚C overnight and secondary antibody incubation was performed at 25˚C for 1 h. The proteins were visualized using ECL procedure (EMD Millipore), and ImageJ v1.8.0 software (National Institutes of Health, Bethesda) was used to analyze the gray intensity of the bands.

Chen L., Lu Q., Chen J., Feng R, & Yang C. (2021). Upregulating miR-27a-3p inhibits cell proliferation and inflammation of rheumatoid arthritis synovial fibroblasts through targeting toll-like receptor 5. Experimental and Therapeutic Medicine, 22(5), 1227.

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Protein Expression Analysis of Tendon Cells

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Total proteins were extracted using pre-cooled RIPA lysis buffer (Beyotime, Beijing, China), then separated by 10% SDS-PAGE gels, followed by shifting onto PVDF membranes. Then, the membranes were probed with primary antibodies at 4 °C for 12 h and then HRP-conjugated antibodies for 2 h at 37 °C. The primary antibodies included Scleraxis (SCX) (1:2000, ab58655), mohawk homeobox (MKX) (1:2000, ab236400), Collagen1A1 (COL1A1) (1:1000, ab34710), Fibromodulin (FMOD) (1:1000, ab267465) and GAPDH (1:100, ab181602) and were obtained from Abcam (Cambridge, MA, USA). The ECL procedure (Merck Millipore) was adopted for proteins observation, and the gray value was evaluated using ImageJ soft (National Institutes of Health, Bethesda, MD, USA).

Yang G., Chen F., Zhang C, & Gu C. (2023). Circ_0005736 promotes tenogenic differentiation of tendon-derived stem cells through the miR-636/MAPK1 axis. Journal of Orthopaedic Surgery and Research, 18, 660.

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Protein Extraction and Western Blot Analysis

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Tissue protein was extracted with a protein extracting reagent (BioTeke Corporation, Beijing, China) according to the manufacturer's directions, while cell protein was extracted through RIPA buffer supplemented with 1% protease inhibitors (Roche, Basel, Switzerland). After centrifugation, the protein content was measured by a BCA Protein Assay Kit (Solarbio, Beijing, China). According to the standard procedures of western blot, total proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA), and then blocked with 5% skim milk in TBST. After being incubated with antibodies, the membranes were visualized with the ECL procedure (Millipore, USA) to get protein bands, which were analyzed by ImageJ software. The primary antibodies included anti-HMGN5, anti-Bcl-2, anti-Bax, anti-Cyclin D1, anti-p21, anti-MMP-2, anti-MMP-9, anti-AKT, anti-p-AKT, anti-ERK1/2, anti-p-ERK1/2 (Santa Cruz Biotechnology, Inc., USA), and anti-β-actin (WanleiBio, Shenyang, China). The secondary antibodies included HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG (OriGene Technologies, USA).

Ma Q., Wang X., Wang H., Song W., Wang Q, & Wang J. (2020). HMGN5 Silencing Suppresses Cell Biological Progression via AKT/MAPK Pathway in Human Glioblastoma Cells. BioMed Research International, 2020, 8610271.

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Western Blot Analysis of Bone Markers

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Total proteins were isolated and separated by 10% SDS-PAGE gels. The proteins were then shifted onto PVDF membranes (Merck Millipore, Billerica, MA, USA). Then the primary antibodies against osteopontin (OPN; ab8448), osteocalcin (OCN; ab93876), SMAD5 (ab40771) and GAPDH (ab8245) were used to incubate with the membranes all night at 4 °C, followed by secondary incubation with HRP-conjugated secondary antibodies for 2 h at 37 °C. All antibodies were obtained from Abcam (Cambridge, MA, USA). Proteins were observed using the ECL procedure (Millipore).

Zheng C., Ding L., Xiang Z., Feng M., Zhao F., Zhou Z, & She C. (2023). Circ_0001825 promotes osteogenic differentiation in human-derived mesenchymal stem cells via miR-1270/SMAD5 axis. Journal of Orthopaedic Surgery and Research, 18, 663.

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Protein Expression Analysis by Western Blot

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Total cellular proteins were extracted and separated by SDS-PAGE gels, and Western blot analysis was performed according to standard procedures. β-actin on the same membrane was used as a loading control. The primary antibodies included anti-CREB (CST, USA), and anti-PCNA, anti-BCL2, anti-CyclinD1, anti-MMP-9 (SantaCruz, USA). Proteins were visualized using the ECL procedure (Millipore, USA). The bands of gray intensity were analyzed by Image J software.

Sun S., Wang X., Xu X., Di H., Du J., Xu B., Wang Q, & Wang J. (2016). MiR-433-3p suppresses cell growth and enhances chemosensitivity by targeting CREB in human glioma. Oncotarget, 8(3), 5057-5068.

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