The total proteins were extracted and the protein concentration was detected using a protein extraction kit and the BCA kit (Thermo Fisher Scientific), respectively. Proteins were insolated on SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific). The membrane was cultured with rabbit polyclonal antibody Bax (1:1000), Bcl-2 (1:1000), Caspase-3 (1:500), E-cadherin (1:1000), β-catenin (1:5000), p-GSK-3β (1:1000), GSK-3β (1:5000), C-myc (1:2000) and β-actin (1:1000) antibodies at 4°C overnight. The next day, the residual primary antibodies were washed with PBST, and the goat anti-rabbit IgG H&L (HRP) antibody (1:5000) was added for incubation at room temperature for 2 hours. The enhanced ECL kit (Thermo Fisher Scientific, USA) was used to measure the OD value, and the protein level was quantified by Image-Pro Plus software. Relative quantification of target protein was carried out with the β-actin as the internal reference. All antibodies were purchased from Abcam (Shanghai) Trading Co., Lld.
Enhanced ecl kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Enhanced ECL kit is a sensitive chemiluminescent detection system for Western blotting. It provides a simple and reliable method for visualizing and quantifying proteins of interest on membrane-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ripa buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Ab97080, by Abcam (1 mentions)
Ab235447, by Abcam (1 mentions)
Ab182858, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Protease phosphatase inhibitor cocktail, by Boster Bio (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Arhgap4, by Abcam (1 mentions)
P akt, by Abcam (1 mentions)
Phospho s6 ribosomal protein ser240 244, by Cell Signaling Technology (1 mentions)
S6 ribosomal protein, by Cell Signaling Technology (1 mentions)
12 protocols using enhanced ecl kit
1
Protein Profiling and Quantification
2
Western Blot Analysis of Chondrocytes
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Cells were harvested and washed with normal phosphate buffered saline (PBS) three times. Then, chondrocytes were lysed in RIPA solutions containing 1% phosphatase/protease inhibitor cocktail (Boster). The bicinchoninic acid (BCA) system was introduced to detect the protein concentration. Afterwards, equal amount of protein samples (30 μg) were electrophoretically separated using 8–12% SDS-PAGE gels, transferred onto PVDF membranes and blocked in 5% BSA at 37 °C for 1 h. Next, the blocked membranes were fully incubated with the targeted primary antibodies at 4 °C for 12 h. Subsequently, the membranes were thoroughly washed with prepared TBST three times and then incubated with corresponding secondary antibodies at room temperature for 1 h. Finally, all bounded protein was visualized by adding enhanced ECL kit (Thermo, USA). The bands were recorded with Bio-Rad scanner device and quantified using Image-J software.
3
Western Blot Protein Analysis Protocol
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The cells were lysed with a commercial Cell Lysis Buffer (#9803, Cell Signalling Technology, Beverly, MA, USA). The protein concentration was determined using a bicinchoninic acid (BCA) assay. The proteins were separated by a SDS-PAGE gel and transferred onto a nitrocellulose (NC) membrane. Blocking was performed with 5% dry milk at room temperature for 2 h. For antibody staining, the membrane was incubated with primary antibody at 4°C overnight, followed by incubation with secondary antibodies at room temperature for 1 h. Protein bands were detected with an enhanced ECL kit (#32016, ThermoFisher Scientific).
4
Protein Extraction and Western Blot Analysis
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Protein extraction from NPCs was carried out with the use of ice-cold RIPA buffer (Beyotime Biotechnology, Shanghai, China). Then, the concentration of the collected protein was assessed by the BCA assay kit (Glpbio, Shanghai, China). Next, proteins were electrophoresed using 12% SDS-PAGE and transferred onto the PVDF membranes carefully. Being blocked with 5% fat-free milk and washed with TBST for 1 h, co-incubation of cells and primary antibody was conducted immediately at 4°C overnight. The primary antibodies used here were against Bcl2, Bax, C-cleaved 3, MMP13, ADAMTS5, Aggrecan, Collagen II, iNOS, COX2, p-P13K, P13K, p-Akt, Akt. Following washing with TBST three times, the membranes were cultured with secondary anti-rabbit antibody in TBST buffer at room temperature for 2 h. An enhanced ECL kit (Thermo Scientific, Waltham, Massachusetts, USA) was applied to visualize the proteins in line with the operating guidelines [24 (link)].
5
Western Blot Protein Analysis
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Cells were lysed in lysis buffer [44 (link)] and cell debris was removed by centrifugation at 14,000xg at 4°C for 15 min. Supernatant extracts were quantified using Bio-Rad protein assay kit. Equal amounts of protein (20-40 μg) were subjected to SDS-PAGE and transferred to PVDF membranes. After transfer, the membranes were incubated with blocking solution and sequentially blotted with primary and second antibodies. The blots were finally developed by the enhanced ECL kit (Thermo Fisher Scientific).
6
Protein Expression Analysis in Ovarian Cancer
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The transfected OVCAR3 and/or SK-OV-3 cells were lysed with RIPA buffer (Beyotime Institute of Biotechnology) to extract total protein. The protein concentration was detected via the BCA Protein Assay kit. Subsequently, a total of 50 µg protein/lane was separated by 10% SDS-PAGE and then transferred onto PVDF membranes. Following blocking with 5% skimmed milk for 2 h at 25˚C, the membranes were incubated overnight at 4˚C with primary antibodies, including anti-TYRP1 (1:1,000; ab235447; Abcam), anti-Bax (1:1,000; ab32503; Abcam), anti-Bcl-2 (1:2,000; ab182858; Abcam) and anti-β-actin (1:1,000; ab265588; Abcam). Thereafter, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; ab97080; Abcam) at 25˚C for 1 h. The proteins bound by their respective antibodies on the immunoblots were measured using an enhanced ECL kit (Thermo Fisher Scientific, Inc.) and quantified using ImageLab software (version 2.3; Bio-Rad Laboratories, Inc.).
7
Western Blot Analysis of Rat Chondrocytes
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Rat chondrocytes were collected and washed with phosphate buffered saline (PBS). Next, the cells were lysed with RIPA buffer containing 1% protease/phosphatase inhibitor cocktail (Boster). The protein concentration of isolated cell lysis solution was determined by bicinchoninic acid (BCA) kit. Afterward, 25 μg protein samples were separated on 8–12% SDS–polyacrylamide gels and transferred to PVDF membranes using a Bio-Rad system. The membranes were blocked with 5% BSA for 1 h at room temperature and then incubated with primary antibodies overnight at 4°C. Subsequently, the membranes were washed with TBST and incubated with corresponding secondary antibodies for 1 h at room temperature. Finally, enhanced ECL kit (Thermo Fisher Scientific, United States) was used to visualize the blots. The relative protein expression was calculated by ImageJ software compared to internal control.
8
Western Blot Analysis of Protein Expression
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Proteins were extracted from lung tissues and BMDMs using RIPA lysis buffer (Sigma-Aldrich, St. Louis, MI, United States) supplemented with PMSF (Beyotime, Shanghai, China). Tissue and cell lysates were centrifuged at 12,000×g for 15 min at 4℃, and then the supernatants were collected. For Western blot assay, proteins were subjected to SDS-PAGE, transferred onto PVDF membranes (Millipore, Bedford, MA, United States), blocked with 5% skim milk for 1 h at room temperature, and subsequently incubated with the indicated primary antibodies overnight at 4℃. The membranes were then incubated with HRP-conjugated anti-rabbit and anti-mouse secondary antibodies for 1 h at room temperature. Subsequently, the bands were visualized with an enhanced ECL kit (Thermo Fisher Scientific, Waltham, MA, United States), and then exposed to the electrochemiluminescence (ECL) system (Tanon, Shanghai, China).
9
Protein Expression Analysis in Poor Ovarian Responders
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Total proteins were extracted and purified from granulosa cells from poor ovarian responders or KGN cells using RIPA reagent (Beyotime Biotechnology). The concentration of purified proteins was analyzed by using BCA Protein Assay Kit (Thermo Fisher Scientific). For western blot, 20 µg of protein was subjected to SDS-polyacrylamide gel, followed by transferring to PVDF membrane (Millipore). The membrane was then subjected to blocking with 5% BSA, then incubation with primary antibodies against ARHGAP4 (1:1000), Bax (1:2000) from Abcam, AKT (1:2000), pAKT (Ser473) (1:1000), S6 Ribosomal Protein (1:2000), Phospho-S6 Ribosomal Protein (Ser240/244) (1:1000), Cleaved Caspase-3 (1:1000) from Cell Signaling Technology and Actin (1:10000) from Sigma-Aldrich, and finally incubation with AffiniPure-conjugated corresponding secondary antibody (Sigma-Aldrich) (1:1000–5000). The targeted protein in membrane was visualized using an enhanced ECL kit (Thermo Fisher Scientific). The expression level of Actin was considered as control.
10
Western Blot Analysis of IgE-Mediated Signaling
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RBL-2H3 cells were seeded into a 60 mm culture dish at 1.0 × 106 cells/5 mL/dish and treated with anti-DNP-IgE for sensitization, followed by treatment with samples and stimulation with DNP-HSA. Fifteen minutes after the stimulation, the cells were washed and lysed using lysis buffer. Cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12.5%) and then transferred onto polyvinylidenefluoride membranes. After blocking for 1 h in blocking buffer (EzBlock Chemi, ATTO Corporation, Tokyo, Japan), the membranes were incubated with a primary antibody at 4 °C overnight, followed by incubation with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The proteins were detected with an enhanced ECL kit (Thermo Fisher Scientific, Waltham, MA, USA) and were visualized by exposing the membrane to a medical X-ray film (Fujifilm Corporation, Tokyo, Japan) in a dark room.
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