The proteins of cartilage or chondrocytes were extracted with RIPA buffer (Beyotime, Shanghai, China) containing 1% phosphatase and protease inhibitors (Bimake, Shanghai, China), followed by measurement of protein concentrations using a BCA protein assay (Beyotime, Shanghai, China). After boiling denaturation, the protein samples (30 μg/sample) were subjected to 10% SDS-PAGE, and then transferred to nitrocellulose membranes (Pall, New York, USA). After being blocked with 5% skim milk for 1 h at room temperature, the membranes were probed with primary antibody at 4 °C overnight and then washed three times with TBS-T before incubation with secondary antibody for 1 h at room temperature. Color development was performed using an ECL chemiluminescence detection kit (Beyotime, Shanghai, China) and images of protein bands were captured using a gel imager (GE, ImageQuant Las4000mini, Japan). GAPDH or β-actin served as the internal control for normalization.
Ecl chemiluminescence detection kit
Manufactured by Beyotime
Sourced in China
The ECL chemiluminescence detection kit is a laboratory equipment designed for the detection and analysis of proteins and biomolecules. It utilizes chemiluminescent reactions to generate light signals that can be measured and quantified, allowing for sensitive and accurate detection of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Imagequant las 4000 mini, by GE Healthcare (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Ripa buffer, by Beyotime (2 mentions)
Bca protein assay, by Beyotime (2 mentions)
Gapdh, by Proteintech (2 mentions)
β actin, by Abcam (2 mentions)
Bcl 2, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor and phosphatase inhibitor, by Selleck Chemicals (1 mentions)
Mmp13, by Abcam (1 mentions)
P ikk, by Cell Signaling Technology (1 mentions)
P nf κb p65, by Cell Signaling Technology (1 mentions)
Sds page, by Solarbio (1 mentions)
Adamts4, by Abcam (1 mentions)
Adamts5, by Abcam (1 mentions)
Mmp 9, by Abcam (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
14 protocols using ecl chemiluminescence detection kit
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Stem Cell Markers
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Total cellular protein from differently treated BMMSCs was isolated by the addition of 1% PMSF and RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS). After boiling in SDS-PAGE sample buffer for 5 min, the samples underwent sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After being blocked for 1 h at room temperature, the membranes were incubated with a 1 : 1000 dilution of the following antibodies (Abs) from Abcam (Cambridge, UK) overnight: Abs against HTERT, SOX2, C-MYC, NANOG, OPN, OCN, RUNX2, PPARγ-2, Lipoprotein lipase (LPL), ADIPONECTIN, and GAPDH. Before detection with an ECL chemiluminescence detection kit (Beyotime Institute of Biotechnology, China), proteins were incubated with horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG (H&L) and HRP-conjugated goat anti-mouse IgG (H&L) secondary antibodies (1 : 5000 dilution) for 1 h at room temperature (Thermo Scientific, USA). The bands were examined with ImageJ software (National Institutes of Health, USA).
3
Protein Extraction and Western Blotting
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Total protein was extracted using RIPA Lysis Buffer (Beyotime, Shanghai, China) containing 1% protease inhibitor and phosphatase inhibitor (Bimake, TX, USA). Equal amounts of protein samples were subjected to 10% or 15% SDS-PAGE (Solarbio, Beijing, China), separated by electrophoresis, and transferred to nitrocellulose filter membranes. The membranes were blocked with 5% skim milk powder for 1 h at room temperature, and then, the target membranes were incubated at 4°C overnight with primary antibodies against COL2A1, ACAN, ADAMTS4, ADAMTS5, MMP3, MMP13, p16 or p21 (1 : 1000, Abcam), ADAMTS5 (1: 250, Abcam), MMP9, iNOS, COX2, NF-κB p65, p-NF-κB p65, AKT, p-AKT, p-PI3K, PI3K, p-IKBα, IKBα, p-IKK or IKK (1 : 1000, Cell Signaling Technology), or GAPDH (1 : 5000, Proteintech). After incubated with HRP-conjugated secondary antibody for 1 h at room temperature, the signal was developed using an ECL chemiluminescence detection kit (Beyotime, Shanghai, China), and images were captured on ImageQuant Las4000mini (GE Healthcare, Tokyo, Japan).
4
Western Blot Analysis of Transcription Regulators
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Total protein of the indicated cells was extracted using RIPA buffer (GenePharma, China), and the protein concentrations were determined using BCA Protein Assay Kit (Beyotime, China). Equal amounts of protein were separated using 10% SDS-PAGE, and transferred to PVDF membrane (Millipore, USA). The membranes were incubated with primary antibodies at 4 °C overnight, followed by incubated with the horseradish peroxidase (HRP)-conjugated secondary antibodies (ab6721, 1:2000, abcam, Cambridge, UK). Finally, protein bands were detected using ECL chemiluminescence detection kit (Beyotime, China). The primary antibodies were as following: anti-KLF12 (ab129459, 1:2000, abcam); anti-ZNF281 (ab101318, 1:2000, abcam); anti-RELA (#3033, 1:2000, Cell Signaling); anti-NRF1 (ab175932, 1:2000, abcam); anti-β-actin (ab8226, 1:2000, abcam).
5
Western Blot Analysis of Signaling Pathways
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Mock-vehicle-treated 97H cells (2.9×106) and overexpressed hsa_circ_0002124-treated 97H cells were treated with pre-cooled cell lysate (volume ratio of PMSF: RIPA cell lysate =1:120). The slurry was centrifuged with 13,000 r/min for 5 min (in a low-temperature mode). Subsequently, the supernatant was transferred to a new 1.5-mL tube, which was immediately stored at −80 °C. The protein loading buffer was added to the collected protein samples. The mixture was heated on a 100 °C boiling water bath for 5 min to completely denature the protein. Thereafter, these proteins were transferred onto a polyvinylidene difluoride membrane (Millipore, USA). After being blocked for 1 h at room temperature, the membrane was incubated with rabbit polyclonal anti-mouse p-JNK (1:2,000), JNK (1:2,000), p-ERK (1:2,000), ERK (1:2,000), p-P38 (1:2,000), P38 (1:2,000), c-Myc (1:2,000), and β-Actin (1:2,000) antibody (BOSTER, USA) for 12 h. Subsequently, the treated proteins were incubated with the corresponding secondary antibody (1:2,000 dilution) for 1 h at room temperature (BOSTER, USA). An ECL chemiluminescence detection kit was used to stain the protein bands (Beyotime Institute of Biotechnology, China), and the Odyssey Infrared Imaging System (LI-COR Biosciences, USA) was used to observe and quantify the protein bands.
6
Protein Extraction and Western Blot
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The SCCVII cells, TEX, BMDCs, DCMV, and Hy‐M‐Exo were first lysed with RIPA buffer to obtain proteins. After denaturation, proteins were then loaded into 10% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE), transferred onto the polyvinylidene fluoride (PVDF) membrane, and probed with certain primary antibodies and secondary antibodies. Finally, an ECL chemiluminescence detection kit (Beyotime Biotech, Shanghai, China) was applied to detect proteins.
7
Western Blot Analysis of Protein Expression
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Western blotting was performed as previously described26 (link). Briefly, cells (1 × 106) were lysed on ice in RIPA buffer containing protease inhibitors. The protein content was measured using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Beijing, China), and total protein (20 μg) was separated by SDS-PAGE electrophoresis, and transferred to PVDF membranes (Millipore Corporation, USA). The membranes were blocked with 5% non-fat milk for 2 h at room temperature and incubated overnight at 4 °C with antibodies, followed by incubation with specific secondary antibodies. Detection of specific proteins was carried out with an ECL chemiluminescence detection kit (Beyotime Institute of Biotechnology, Beijing, China) according to the manufacturer’s instruction. The western blot images were obtained with an MiniChemi 610 chemiluminescent imager (Sagecreation, Beijing, China). All experiments were performed at least in triplicate, and quantitative analysis of blots were performed using the Fiji based ImageJ software (version 1.51n, National Institute Health, Bethesda, MA, USA).
8
Western Blot Analysis of Apoptosis Markers
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Cellular proteins were extracted using Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime, Shanghai, China) and quantified using the Bicinchoninic acid (BCA) Protein Assay Kit (Beyotime). Next, 50 μg of proteins was loaded and separated on 12% or 15% gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were transferred onto polyvinylidenefluoride (PVDF) membranes (Millipore, Billerica, MA, USA) at room temperature. The membranes were blocked in 5% skim milk in TBST for 1 h and incubated with primary antibodies against Bcl-2 (12789-1-AP, 1:1000, Proteintech, Wuhan, China), Bax (50599-2-lg, 1:1000, Proteintech), and GAPDH (ab9485, 1:2,500, Abcam) overnight at 4°C. The membranes were blocked with the appropriate anti-mouse IgG antibody (ab6728, 1:3000, Abcam) at room temperature for 1 h. Resultant bands were analyzed using an ECL chemiluminescence detection kit (Beyotime).
9
Western Blot Analysis of HEK293T Proteins
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The proteins of HEK293T cells were extracted with RIPA buffer (Beyotime) containing 1% phosphatase and protease inhibitors (Bimake), followed by measurement of protein concentrations using a BCA protein assay (Beyotime). After boiling denaturation, the protein samples (20 μg/sample) were subjected to 7.5% SDS‐PAGE and then transferred to nitrocellulose membranes (Pall). After being blocked with 5% skim milk for 1 hr at room temperature, the membranes were probed with primary antibody against Flag (101274‐MM05, Sino Biological), HA (100028‐MM10, Sino Biological) at 4 °C overnight and then washed three times with TBS‐T before incubation with antimouse secondary antibody (7076, Cell Signaling Technology) for 1 hr at room temperature. Color development was performed using an ECL chemiluminescence detection kit (Beyotime, Shanghai, China), and images of protein bands were captured using a gel imager (GE, ImageQuant Las4000mini).
10
Immunoblotting Analysis of Key Proteins
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Immunoblotting was performed according to standard procedures. The following primary antibodies against VEGFA (1:1000, #66828‐1‐Ig; Proteintech Corp.), CD9 (1:1000, #13403S; Cell Signaling Technology, Danvers, MA), HIF1α (1:250, #36169; Cell Signaling Technology), SOX9 (1:1000, #ab185966; Abcam), RUNX2 (1:1000, #12556s; Cell Signaling Technology), and GAPDH (1:5000, #10494‐1‐AP; Proteintech Corp.) were used. After incubation with HRP‐conjugated secondary antibodies for 1 h at room temperature, the signals were developed using an ECL chemiluminescence detection kit (Beyotime Biotechnology, Beijing, China) and images were captured on an ImageQuant Las4000 mini system (GE Healthcare, Chicago, IL).
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