After 24 h drug treatment, total proteins were extracted from cells with RIPA lysis buffer and measured using the bicinchoninic acid protein assay kit (Biyuantian Inc., China). Protein lysates were separated by denaturing SDS-PAGE and analyzed by WB with designated primary and secondary antibodies as per standard protocol. Signals were detected using the ECL Plus Kit (Biyuntian Inc., China).
Ecl plus kit
Manufactured by Beyotime
Sourced in China, United States
The ECL Plus kit is a laboratory equipment used for chemiluminescent detection of proteins in Western blotting analysis. It provides a sensitive and reliable method for visualizing target proteins on a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (20 mentions)
Bca protein assay kit, by Beyotime (15 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Ripa lysis buffer, by Beyotime (9 mentions)
Ripa buffer, by Beyotime (6 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
β actin, by Abcam (4 mentions)
Ipvh00010, by Merck Group (4 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (4 mentions)
Canoscan lide 100 scanner, by Canon (3 mentions)
Ab8227, by Abcam (3 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
Bicinchoninic acid kit, by Beyotime (3 mentions)
Mmp 9, by Abcam (2 mentions)
Anti β actin, by Abcam (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Ethidium bromide, by Merck Group (2 mentions)
Bca kit, by Beyotime (2 mentions)
Polyvinylidene di uoride, by Merck Group (2 mentions)
75 protocols using ecl plus kit
1
Protein Extraction and Western Blot
2
HMGB2 Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were extracted with RIPA lysis buffer (Biyuntian, Hangzhou, China). Protein lysates were then separated by 10% sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with the primary antibodies HMGB2 (14597–1-APl, Proteintech) and β-actin (Abcam, ab133626, 1:5000) overnight at 4 °C. The membranes were then incubated in HRP-linked secondary antibodies (anti-rabbit IgG; Cell Signaling Technology, Danvers, MA, USA; 1:7500) for 2 h. Western blotting signals were detected using the ECL Plus kit (Biyuntian). Each experiment was repeated 3 times independently.
3
Western Blot Analysis of Stem Cell Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed for 30 min on ice in RIPA lysis buffer (Solarbio, Beijing, China) containing phenylmethylsulfonyl fluoride (1,000:1) and centrifuged to collect the supernatants. The protein concentration in each supernatant was determined using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Protein lysates were incubated in a 95 °C water bath for 10 min in Lithium Dodecyl Sulfate (LDS) sample buffer (4×), and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Solarbio, Beijing, China). After transfer, the membranes were blocked with 7% skim milk for 60 min at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti-SOX2 (1:1,000, Abcam, Cambridge, MA, USA), anti-OCT4 (1:1,000, Abcam), and anti-β-actin (1:1,000, Abcam). The membranes were washed and incubated with secondary antibodies for 90 min at room temperature. Proteins were visualized using the ECL Plus Kit (Biyuntian, China) and analyzed using image analysis software (ImageJ, version 1.48; National Institutes of Health, Bethesda, MD, USA).
4
Investigating Cellular Signaling Pathways in Treated Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells after treatment, were extracted with RIPA lysis buffer (Biyuntian, Hangzhou, China). Protein lysates were then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with the primary antibodies: P21 (1:1,000), Ki-67 (1:1,000), PCNA (1:1,000), MMP-2 (1:1,000), MMP-9 (1:2,000), FOXM1 (1:1,000) and β-actin (1:1,000) (all from Abcam, Cambridge, MA, USA) overnight at 4°C. Then, the membranes were incubated in HRP-linked secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h. Western blotting signals were detected using the ECL Plus kit (Biyuntian). Each experiment was repeated 3 times, independently.
5
Western Blot Analysis of Extracellular Matrix Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein from cells was extracted with radio immunoprecipitation assay (RIPA) lysis buffer (Biyuntian, China). Protein lysates were then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (PVDF, Millipore, Massachusetts, USA). After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with the primary antibodies: MMP-2 (1:1000, Abcam, USA), MMP-9 (1:1000, Abcam, USA), TIMP-1 (1:1000, Abcam, USA), TIMP-2 (1:2000, Abcam, USA), SOX4 (11000, Abcam, USA), and β-actin (1:1000, Abcam, USA) overnight at 4°C. Then the membranes were incubated in horseradish peroxidase (HRP)-linked secondary antibodies (Santa Cruz Biotechnology, USA) for 2 h. Western blotting signals were detected using the ECL Plus Kit (Biyuntian, China). Each experiment was repeated three times independently.
6
CSFV-Stimulated Cell Lysis and Western Immunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western and immunoprecipitation (IP) cell lysis buffer (P0013; Beyotime) was chilled on ice, and phenylmethylsulfonyl fluoride (PMSF) purchased from Beyotime (ST506) was added to a final concentration of 1 mM prior to use. Cell lysates were recovered from CSFV-stimulated or control samples via cell lysis for 45 min on ice. The lysate was centrifuged at 13,000 × g for 25 min at 4°C. Next, the supernatant was collected and the protein concentration measured using a BCA protein assay kit (23227; Thermo Fisher Scientific). Protein samples were boiled for 5 min in 5 × sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. Equal amounts of protein for each sample were separated using 12% SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF) membranes (IPVH00010; Millipore). The PVDF membranes were blocked with 5% skim milk dissolved in PBST at 37°C for 2 h. Next, the PVDF membranes were incubated with primary antibodies at 4°C overnight, and the corresponding HRP-conjugated secondary antibodies were added at 37°C for 1 h, at appropriate dilutions (1:1,000). An ECL Plus kit (P0018; Beyotime) and chemiluminescence imaging system (Fine-do X6, Tanon) were used for detecting and imaging the protein bands. Image-Pro Plus 6.0 software (Media Cybernetics) was used to quantitate the protein in the blots according to the user's guide.
7
Western Blot Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysis was conducted in RIPA lysis buffer, and then the lysate was separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore). Blocked with 5% nonfat milk, the membranes were incubated with primary antibodies overnight at 4 °C. After an incubation with secondary antibody at 37 °C for 1 h, enhanced chemiluminescence (ECL) Plus kit (Beyotime, Shanghai, China) visualized the protein bands.
8
Western Blot Protein Detection Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blots were performed as previously described (10 (link)). Protein bands were detected by using the ECL Plus kit (P0018;
Beyotime Biotechnology, Jiangsu, China), and images were obtained by using a CanoScan
LiDE 100 scanner (Canon, Tokyo, Japan). Protein blots were analyzed by using ImageJ
software (National Institutes of Health, Bethesda, MD, USA).
Beyotime Biotechnology, Jiangsu, China), and images were obtained by using a CanoScan
LiDE 100 scanner (Canon, Tokyo, Japan). Protein blots were analyzed by using ImageJ
software (National Institutes of Health, Bethesda, MD, USA).
9
Western Blot Protocol for Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All the manipulation was performed as previously described.22 (link) Total proteins were extracted from fresh tissues by RIPA lysis buffer (KeyGEN, China) and measured by BCA Protein Assay Kit (KeyGEN). A total of 50 μg of protein was separated on 10% SDS-polyacrylamide gels (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocked with 5% nonfat milk PBST solution for 1 hr at room temperature, membranes were incubated with polyclonal RRBP1 antibody (1:1,000) (Proteintech, China) and β-actin antibody (1:2,000) (Abcam, USA) at 4 °C overnight, respectively. Then, membranes were washed with PBST and incubated with secondary antibodies (ZhongshanJingqiao, China) for 1 hr at room temperature. Protein band was visualized by ECL Plus Kit (Beyotime, China). Relative protein expression levels were statistically analyzed by measuring the gray density of each protein band. The expression of β-actin was used as an internal control protein for standardizing the expression of target protein. Experiments were repeated in triplicate.
10
Comprehensive Cell Viability Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RPMI-1640 medium (Gibco, USA), fetal bovine serum(FBS, Gibco, USA), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, Fluka, USA), acridine orange (AO, Amresco, USA), ethidium bromide (EB, Sigma, USA), Wright-Giemsa dye solution (Nanjing Jiangcheng Bioengineering Institute, Nanjing, China), RNase A (Fermantas, Canada), proteinase K (Fermantas, Canada), ECL plus kit (Beyotime Institute of Biotechnology, China), DL 2000 DNA Marker (Takata, Japan), prestained protein marker (fermentas, Thermo Scientific, USA), antibody to GAPDH, GRP78, GADD153, Bcl-2, Bax, p53, survivin, Actived-Caspase 3 p17 (Bioworld Technology, MN, USA), horseradish peroxidase (HRP)-conjugated secondary antibody (MultiSciences, China). All other biochemicals and chemicals used in the experiment were of analytical grade.
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!