Cells in culture flasks or plates were lysed in ice‐cold buffer containing protease inhibitor cocktail (Roche). Equal amounts of protein samples were loaded and separated by SDS‐PAGE and transferred to PVDF membranes (Millipore) followed by non‐fat milk or BSA blocking. These blots were incubated at 4°C overnight with primary antibodies against ZEB2 (1:1000 dilution; Proteintech, 14026‐1‐AP), Cleaved‐PARP (1:1000 dilution; Cell Signaling, #9541), N‐cadherin (1:1000 dilution; Cell Signaling, #13116), E‐cadherin (1:1000 dilution; Cell Signaling, #14472), Vimentin (1:1000 dilution; GeneTex, GTX100619), Snail (1:1000 dilution; Cell Signaling, #3879) and GAPDH (1:5000, MB001, Bioworld) followed by incubation with horseradish peroxidase (HRP)‐conjugated secondary antibodies at room temperature. Immunoreactive bands on the blots were detected by ECL chemiluminescence kit (Millipore).
Ecl chemiluminescence kit
Manufactured by Merck Group
Sourced in United States, Germany
The ECL chemiluminescence kit is a laboratory equipment product designed for the detection and quantification of proteins in Western blot analysis. The kit utilizes a chemiluminescent substrate to generate a luminescent signal upon reaction with the target protein, allowing for sensitive and accurate protein visualization and measurement.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (22 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
β actin, by Santa Cruz Biotechnology (7 mentions)
Ripa lysis buffer, by Beyotime (6 mentions)
Chemidoc mp imaging system, by Bio-Rad (6 mentions)
Polyvinylidene fluoride membrane, by Merck Group (6 mentions)
Ripa lysis buffer, by Boster Bio (6 mentions)
Bca kit, by Thermo Fisher Scientific (6 mentions)
Ripa buffer, by Beyotime (4 mentions)
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
Image pro plus software, by Media Cybernetics (4 mentions)
Chemidoc xr, by Bio-Rad (3 mentions)
Gel pro analyzer 6, by Media Cybernetics (3 mentions)
Ripa lysate, by Beyotime (3 mentions)
Ab205718, by Abcam (3 mentions)
N cadherin, by Cell Signaling Technology (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Vimentin, by GeneTex (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
77 protocols using ecl chemiluminescence kit
1
Western Blot Analysis of EMT Markers
2
Western Blot Analysis of Protein Levels
3
Immunoblotting Protein Detection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The preparation of total cell extracts and immunoblotting with appropriate antibodies were performed as previously described.37 (link) The appropriate antibodies were used as indicated in Supplementary Data . Labeled proteins were visualized with an ECL chemiluminescence kit (Millipore).
4
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells specimens were lysed using RIPA lysis buffer (Boster, Wuhan, China). The equal amounts of total proteins were separated by 10% SDS-PAGE gel and then transferred onto a PVDF membrane (Millipore, MA, USA). The membranes were blocked with 5% skim milk powder at room temperature for 1 h and incubated with primary antibodies diluted in blocking buffer at 4°C overnight. Subsequently, the membranes were washed and incubated with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were detected by an ECL chemiluminescence kit (Millipore) according to the manufacturer's instructions. Protein levels were calculated relative to β-actin. Primary antibodies were used as follows: rabbit anti-NFAT2 (phospho S237) (Abcam, Cambridge, UK, ab183023), rabbit anti-p53 (#2527), rabbit anti-Bax (#5023), rabbit anti-Bcl-2 (#4223), rabbit anti-cleaved-caspase-3 (#9664), rabbit anti-NFAT2 (#8032), and rabbit anti-β-actin (#4970) were all from cell signaling (Danvers, MA, USA).
5
Western Blot Analysis of Apoptosis Regulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RIPA buffer (Biossci, Wuhan, China) was added to the cells in each group. After fully mixed, the lysis was incubated on ice for 30 min. Then, the lysis was centrifuged for 15 min at 12,000 r/min at 4°C, and the supernatant was collected. After SDS-PAGE, the proteins were transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA) and afterwards the membranes were blocked with 5% skimmed milk. Then, the membranes were incubated at 4°C overnight with primary antibodies anti-Bcl-2, Bax, MMP11 and β-actin. After washing the membranes with TBST, they were incubated with HRP-labeled secondary antibodies for 1 h at room temperature. Subsequently, equivoluminal liquid A and liquid B of the hypersensitive ECL chemiluminescence kit (Millipore, Bedford, MA, USA) was mixed and cover the PVDF membranes to react with antigen-antibody complex. Then, the protein bands were scanned. The antibodies used in this study included anti-MMP11 antibody (ab53143, 1:2000), anti-Bcl-2 antibody (ab185002, 1:2000), anti-Bax antibody (ab32503, 1:2000) and anti-β-actin antibody (ab179467, 1:2000), which were all available from Abcam (Shanghai, China).
6
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted in cold by using RIPA lysis buffer (Beyotime Biotechnology, China), and the protein concentration was measured by the BCA Assay Kit (Thermo Scientific). Protein fractions were separated with sodium dodecyl sulfate/polyacrylamide gel electrophoresis gels (10%) and then transferred on to polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, U.S.A.). After blocking with 5% BSA for 2 h at 4°C, membranes were incubated with specific primary antibodies overnight at 4°C. Anti-Bax, anti-caspase-3 and anti-GAPDH were purchased from Cell Signaling Technology (Boston, MA, U.S.A.). The membranes were washed thrice and then incubated with secondary antibodies for 1 h. The protein bands were visualized using an ECL Chemiluminescence kit (Millipore, Billerica, MA, U.S.A.). GAPDH was considered as the internal control.
7
Protein Expression Profiling by Western Blot
8
Protein Extraction and Immunoblot Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein extraction and immunoblot assay were performed as previously described [16 (link)]. Briefly, cells were washed with 1x PBS and resolved in RIPA buffer (100 mM Tris, 5 mM EDTA, 5% NP40; pH 8.0) with protease inhibitors (1 mM phenyl-methyl sulphonyl fluoride, 1 μg/mL aprotinin, 1 μg/mL leupeptin). Proteins were resolved by SDS-PAGE and then transferred to PVDF membranes. Blocking of nonspecific binding was accomplished by adding 5% nonfat milk. Primary antibodies were applied and then incubated overnight at 4°C. Secondary antibodies were then added and incubated. Signals were enhanced using an ECL chemiluminescence kit (Millipore, US) and detected by ChemiDoc XRS+ (BioRad).
9
Western Blot Analysis of Protein Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell pellets were lysed by SDS or RIPA solution incorporating an inhibitor of proteinase (Cell Signaling Technology, USA), and total protein was extracted immediately following the manufacturer's instructions. Approximately 20 μg of protein was loaded and separated on NuPAGE bis-tris mini gels (Invitrogen) or SDS polyacrylamide gels and subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). Then, the PVDF membranes were blocked after treatment with 5% non-fat milk at room temperature for 1 h. Blocked membranes were mixed with a solution of specific primary antibodies at 4°C overnight. Subsequently, secondary antibodies (Proteintech, China) were added and reacted with the primary antibodies at room temperature for 1 h. After staining via ECL chemiluminescence kit (Millipore), protein bands were detected by a ChemiDoc MP imaging system (Bio-Rad, USA). Table S4 shows the antibodies used for western blot.
10
Protein Extraction and Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the extraction of proteins from the cell lines, cell lysis buffer containing protease inhibitors was used. The extracted protein concentration was measured using the BCA Protein Assay Kit (Thermo Scientific). For the western blot analysis, 20 μg of protein extracts from each clone was loaded onto 7.5% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes (Roche Diagnostics). After blocking, the protein was probed with different antibodies (shown in the Supplementary file) and an anti-α-tubulin antibody was used as a loading control. Signals were detected using an ECL chemiluminescence kit (Millipore Corporation, Billerica, MA, USA). Total RNA was isolated using the TRIzol reagent (Life Technologies Corporation, Carlsbad, CA, USA) according to the manufacturer’s recommendations. The 2−ΔΔCt method of relative quantification was used to estimate the copy number of the expression of each gene, and 18 S was selected as the internal control. All of the primers used in the study are listed in the Supplementary file.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!