Cells on 6-well plates were rinsed twice with cold PBS and lysed in RIPA lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% deoxycholicphenyl methylsulfonyl fluoride, 5.0 mM sodium pyrophosphate, 1.0 g/mL leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, and1 mM DTT) containing a protease inhibitor (PMSF) mixture at 1:100 dilution on ice for 30 min. The insoluble components of cell lysates were removed by centrifugation (4 oC, 12000 × g, 10 min), and protein concentrations were measured using a Pierce BCA protein assay kit. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked using 5% skim milk in TBST and then incubated with diluted indicated primary antibodies: anti-GPX4 (1:1000 dilution, Proteintech, Cat No: 30388-1-AP), anti-SLC7A11 (1:1000 dilution, ABclonal, Cat No: A13685), anti-GAPDH (1:1000 dilution, Wanlei, Cat No: WL01114) at 4 oC with gentle shaking overnight. After washing five times (5 min each) with TBST, membranes were incubated with the HRP conjugated goat anti rabbit IgG (H + L) (1:5000 dilution, Wanlei, Cat No: WLA023) for 1 h at rt. Immunoreactive bands were visualized using an ECL detection kit (Wanlei, Cat No: WLA006) following the manufacturer’s instruction on the ChemiDoc system (BioRad, Shanghai, China).
Ecl detection kit
Manufactured by Wanlei
Sourced in China
The ECL detection kit is a laboratory equipment designed to facilitate the detection of target molecules using the enhanced chemiluminescence (ECL) technique. This kit provides the necessary reagents and components to perform ECL-based detection, which is a widely used method in various biochemical and molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Wl0088, by Wanlei (2 mentions)
Ripa buffer, by Wanlei (2 mentions)
Anti gpx4, by Proteintech (1 mentions)
Anti slc7a11, by ABclonal (1 mentions)
Anti gapdh, by Wanlei (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Wl03358, by Wanlei (1 mentions)
Bca protein concentration determination kit, by Wanlei (1 mentions)
Wl01506, by Wanlei (1 mentions)
Wl01845, by Wanlei (1 mentions)
Goat anti rabbit igg hrp, by Wanlei (1 mentions)
Anti smad2 3, by Affinity Biosciences (1 mentions)
Bca protein quantification kit, by Beyotime (1 mentions)
Avastin, by Roche (1 mentions)
A00702, by GenScript (1 mentions)
A1483, by ABclonal (1 mentions)
Wl01435a, by Wanlei (1 mentions)
P0013, by Beyotime (1 mentions)
7 protocols using ecl detection kit
1
Western Blot Analysis of Ferroptosis Markers
2
Western Blot Analysis of Bone and Matrix Proteins
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Cells were harvested and lysed with lysis buffer containing the protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Each group of protein samples was quantified using the BSA Protein Quantification kit. Equal amounts of protein (20 µg/lane) from the sample were electrophoresed on 10% SDS-PAGE gels and were transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBS containing 0.1% Tween-20 for 1 h at room temperature. After washing with TBST three times, the membranes were co-incubated with the primary antibody (1:500 for anti-BSP, 1:500 for anti-MMP2, 1:500 for GAPDH) overnight at 4°C in TBST. After incubation with horseradish peroxidase (HRP) goat anti-rabbit IgG (1:10,000) in TBST for 60 min, the proteins were visualized using the ECL detection kit (Wanleibio). Imaging system used ImageQuant™ LAS 4000 (GE Healthcare, Chicago, IL, USA).
3
Western Blot Analysis of Autophagy Markers
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The cells were lysed with Ripa buffer (wanleibio, China) containing 10 mM protease inhibitor (PMSF; wanleibio, China). The lysate was centrifuged at 12,000 rpm and 4 °C for 10 min. The precipitation was separated and the protein concentration was determined by the BCA protein concentration determination kit (wanleibio, China). The equivalent protein (40 μg) was added to the 8–15% SDS-PAGE gel and then transferred to the PVDF membrane (Millipore, Billerica, USA). The membrane was sealed in 5% skimmed milk powder solution for 1 h, and then incubated overnight at 4 °C with antibodies specific for LC3 (1:500; wl01506; wanleibio, China), runx2 (1:500; wl03358; wanleibio, China), collagen I (1:500; wl0088; wanleibio, China), β-actin (1:1000; wl01845; wanleibio, China). After washing 4 times with TBST, the membrane was incubated with goat anti-rabbit IgG-HRP (1:5000; WLA023; wanleibio, China) for 45 min at room temperature. The protein bands were visualized using ECL detection kit (wanleibio, China) and compared on the same membrane. We used gel image processing system (Gel-Pro-Analyzer software) to analyze the optical density of target strips and calculate the proportion of LC3-II/I in order to determine the level of autophagy.
4
TGF-β1 Signaling in Wound Healing
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Bev injection (100 mg/4 mL, Avastin, Roche Pharma, Switzerland);Lis hydrochloride tablets (10 mg, Zhongfu, China); 5-Fu injection (0.25 g/mL, Xudong, China); 0.9% sodium chloride injection (100 mL, Baxter, China);Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) (HyClone, UT, USA); a BCA protein quantification kit (Beyotime Biotechnology, Shanghai, China); an ECL detection kit, anti-TGF-β1 and anti-Collagen I (Wanleibio, Shenyang, China); anti-Smad2/3, anti-phospho-Smad2/3 and anti-Smad4 (Affinity Biosciences, OH, USA); anti-CD31 and anti-α-SMA (Servicebio, Wuhan, China); anti-GAPDH and secondary antibodies for Western blot analysis (Proteintech, Chicago, USA) were used.
5
Protein Extraction and Western Blot
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Total protein was extracted using the sodium dodecyl sulfate (SDS) lysis buffer (1% sodium dodecyl sulfate, 5% glycerol, 1 mM ethylenediamine tetraacetic acid (EDTA), 25 mM Tris, 150 mM NaCl, and 1 mM phenylmethylsulfonyl fluoride (PMSF)). Protein samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 5% non-fat milk powder and incubation with specific antibody at 4°C overnight, membranes were subjected to corresponding secondary antibody and then visualized by an ECL detection kit (Wanleibio, Dalian, China).
6
Immunoblot Analysis of Cell Cycle Regulators
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Total proteins were extracted using cell lysis buffer (P0013, Beyotime) and separated by SDS‐PAGE. Then, proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). After blocking with 5% skim milk, the membranes were incubated with primary antibodies against SHMT1 (1:1000, D3B3J, CST), cyclinB (1:1000, 4138S, CST), CDC20 (1:1000, 4823S, CST), β‐actin (1:1000, A00702, GenScript), HOXD8 (1:1000, sc‐515357, SantaCruz), p21 (1:1000, A1483, ABclonal), γ‐H2AX (1:500, CSB‐PA010097OA139phHU, CUSABIO), p‐CDK1 (1:1000, CSB‐PA000492, CUSABIO), p‐CHK1 (1:1000, CSB‐PA006769, CUSABIO), p‐CDC25 (1:1000, CSB‐PA080107, CUSABIO), cyclinE (1:1000, sc‐377100, SANTA), and cyclinD (1:1000, WL01435a, WanleiBio) overnight at 4°C with shaking. Then, the membranes were washed with Tris Buffered Saline with Tween20 (TBST) and incubated with secondary antibody conjugated with HRP (1:5000, GenScript) for 1 h at room temperature. The signals were developed by the ECL detection kit (Wanleibio).
7
Western Blot Analysis of Osteogenic Markers
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Cells were lysed with Ripa buffer (Wanleibio, China) containing 10 mM protease inhibitor (PMSF; Wanleibio, China). The lysate was centrifuged at 12,000 rpm and 4°C for 10 min. The supernatant was separated, and the protein concentration was determined using a BCA protein concentration assay kit (Wanleibio, China). Equivalent amounts of protein (40 g) were added to the 8%-15% SDS-PAGE gel and then transferred to a PVDF membrane (Millipore, Billerica, United States). The membrane was sealed in 5% skimmed milk powder solution for 1 h, and then incubated with the following primary antibodies: runt-related transcription factor 2 (Runx2, wl03358; Wanleibio, China), Collagen I (wl0088; Wanleibio, China), β-actin (wl01845; Wanleibio, China), CD9 (ab92726, Abcam, United Kingdom), CD63 (ab216130, Abcam, United Kingdom), and TSG101 (ab125011, Abcam, United Kingdom) at 4°C overnight. After washing with TBST four times, the membrane was incubated with sheep anti-rabbit IgG HRP (wla023; Wanleibio, China) at room temperature for 45 min. An ECL detection kit (Wanleibio, China) was used to visualize the protein bands. Proteins on the same membrane were compared quantitatively by determining the optical density of the target strip using a gel image processing system (Gel-Pro-Analyzer software, Media Cybernetics, United States).
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