Total protein was extracted from cells using Beyotime Cell Lysis Buffer for Western and IP (Beyotime, China) containing protease inhibitors. Proteins were separated by 8% SDS-PAGE and transferred to a methanol-activated PVDF membrane. The membrane was blocked for 1 h in 5% nonfat milk and subsequently probed with an antibody against human c-MYC (1:1500; Cell Signaling Technology, USA) at 4 °C overnight. β-Actin served as a loading control (1:1500; Beyotime, China). Membranes were washed three times for 5 min each with phosphate-buffered saline with 0.05% Tween-20 (PBST) and were incubated with goat anti-rabbit or anti-mouse IgG (1:2500; Beyotime, China) for 1 h, washed three times with PBST and detected with Super ECL Detection Reagent (Shanghai YEASEN Biotechnology Co. Ltd, China). Bands were imaged by the Tanon 5200 Chemiluminescent Imaging System (Tanon, China).
Super ecl detection reagent
Manufactured by Yeasen
Sourced in China
The Super ECL Detection Reagent is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. It produces a robust and sustained luminescent signal, allowing for sensitive and quantitative detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (15 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Image lab software, by Bio-Rad (3 mentions)
Ripa lysis buffer, by Solarbio (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Solarbio (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Amersham imager 600, by Cytiva (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
High sensitivity ecl chemiluminescence detection kit, by Vazyme (2 mentions)
Lysis buffer, by Beyotime (2 mentions)
Anti gapdh, by Proteintech (2 mentions)
Polyvinylidene fluoride membrane, by GE Healthcare (2 mentions)
Cell counting kit 8 cck 8, by Targetmol (2 mentions)
Bca protein quantification kit, by Yeasen (2 mentions)
Gapdh, by Proteintech (2 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
54 protocols using super ecl detection reagent
1
Western Blot Analysis of c-MYC
2
Exosomal Protein Characterization by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The extracted sEVs were lysed in RIPA buffer (Solarbio, China), boiled, and loaded onto a 10% Tris–Gly gel. The separated proteins were transferred to polyvinylidene difluoride membranes, which were subsequently blocked with 5% skim milk in TBST for 60 min at room temperature. After three washes, the membrane was incubated overnight at 4 °C with primary antibodies against CD63 (ab134045, Abcam), CD81 (ab109201, Abcam), TSG101 (DF8427, Affinity Biosciences), Calnexin (610,523, Becton Dickinson Company), complement C1Q subcomponent subunit B (C1QB; 16919-1-AP, Protein Tech), complement C1Q subcomponent subunit C (C1QC; 16889-1-AP, Proteintech), C4B-binding protein alpha chain (C4BPA; 11819-1-AP, Proteintech), and C4B-binding protein beta chain (C4BPB; 15837-1-AP, Proteintech). After washing, the membrane was incubated for 60 min at room temperature with an HRP-conjugated secondary antibody (Ray, China) and then detected using a Super ECL detection reagent (Yeasen, China). The immunoreactive bands were visualized using a Tanon Automatic Chemiluminescence and Fluorescence Image Analysis System (Tanon, China).
3
Western Blot Analysis of TREM2 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Brain tissues or leukocytes were lysed in 1% Triton X-100 lysis buffer (Solarbio, Beijing, China) supplemented with protease and phosphatase inhibitor cocktails (Thermo Scienti c,USA). The protein concentration was determined using a BCA Protein Assay kit(Thermo Scienti c, USA). Equal amounts of protein were loaded onto SDS-PAGE gels (with 10% SDS) and then were separated by electrophoresis. The seperated proteins were then transferred to PVDF membranes (Millipore, California, USA). The membranes were blocked with 5% w/v nonfat milk for 1 hour at room temperature. The membranes were then incubated overnight at 4 °C with a TREM2 antibody (1:500 dilution; Abcam) and a β-actin antibody (1:5000 dilution; ZSGB-BIO) in TBST containing 5% w/v nonfat milk. The cells were washed 3 × 5 minutes in TBST at room temperature. The membranes were incubated for 1 hour at room temperature with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (ZSGB-BIO, Beijing, China) in TBST containing 5% w/v nonfat milk. After extensive washing in TBST the protein bands were visualized using an Super ECL Detection Reagent (YEASEN,Shanghai,China) and then were imaged using a Tanon 4600SF system (China).
4
Western Blot Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Phenylmethanesulfonyl fluoride (PMSF) (1mM) was added to the prechilled radioimmunoprecipitation assay (RIPA) buffer to obtain cell lysates, and these lysates were used to acquire the total proteins. The protein concentrations were quantified by a bicinchoninic acid (BCA) Protein Assay Kit (Beyotime, Nantong, China). Protein separation was conducted through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Burlington, MA, USA). 5% skim milk in Tris-buffered saline-Tween (TBST) was used to block the membranes at ambient temperature for 2 h. Then, the membranes were incubated with diluted primary antibodies at 4°C overnight, followed by three washes with TBST. The membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at 37°C, washed with TBST, and visualized by the Super ECL Detection Reagent (Yeasen, Shanghai, China). The protein density was then quantified using Image Lab software (Bio-Rad, Hercules, CA, USA). The antibodies used in this study are listed in Supporting Table 2
5
Western Blot Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins are detected by Western blotting. After incubation, cell lysates were heated in 1x SDS loading buffer for 15 min at 100 °C. Proteins were separated by SDS-PAGE gel, after being transfered to 0.45 μm polyvinylidene fluoride membranes, blocked with 5% BSA for 1 h. Membranes were incubated with antibodies against cyclinB1, cyclinA2, CDC2, P21, PARP, p-JNK, and GAPDH at 4 °C overnight. Secondary antibody was incubated for 1 h. Bands were detected by a chemiluminescence imaging system (Amersham Imager 600) with Super ECL Detection Reagent (Yeasen, Shanghai, China). Results were analyzed by Image J.
6
Protein Extraction and Immunoblotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lysis buffer (Beyotime) supplemented with 1% phenylmethylsulfonyl fluoride (Beyotime) was used to extract total protein from cells. Cell extracts were centrifuged at 12,000g for 15 min, and the supernatant was collected. Cell lysates were resolved on 8% acrylamide gradient SDS–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% nonfat milk in tris-buffered saline containing 0.1% Tween 20 for 1 hour, then incubated with anti-BRD4 (1:1000 dilution; Abcam) and anti–β-actin (1:10000 dilution; Sungenebiotech) antibody followed by horseradish peroxidase–conjugated secondary antibody, and detected by immunoblotting with the High-Sensitivity ECL Chemiluminescence Detection Kit (Vazyme) or Super ECL Detection Reagent (Yeasen Biotech).
7
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from liver tissue samples or cells using RIPA lysis buffer (Solarbio, Beijing, China). Then, the final liquid supernatants were harvested via centrifugation at 13,500 rpm for 30 min. Protein concentrations were calculated using PierceTM Rapid Gold BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Next, the protein samples were subjected to 10% or 12% SDS-PAGE and transferred into PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked in 5% nonfat milk, then incubated with specific primary antibodies at 4 °C overnight. The membranes were incubated with HRP-conjugated secondary antibody. After washing, protein bands were visualized using Super ECL Detection Reagent (Yeasen Biotech Co., Ltd., Shanghai, China) and exposed to Kodak (Eastman Kodak Company, Rochester, NY, USA) X-ray film. Corresponding protein expression was determined as a gray value (ImageJ, Version 1.4.2b, National Institutes of Health, Bethesda, MA, USA), standardized to the housekeeping gene (GAPDH), and expressed as a fold of the control.
8
Protein Expression Analysis in GC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GC tissues and cells were lysed in ice-cold RIPA buffer with 1 mM PMSF. Total protein was separated by SDS-PAGE, transferred to PVDF membranes (Millipore, Bedford, MA, USA) and blocked with 5% skim milk in TBST for 2 h. Then, the membranes were incubated with primary antibodies specific for ASCT2, GLS1, USP5, β-catenin, c-Myc and GAPDH at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 2 h. The signals were visualized with Super ECL Detection Reagent (Yeasen, Shanghai, China), and images were acquired using Image Lab software (Bio-Rad, Hercules, CA, USA).
9
RNA m6A Dot Blot Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Dot blot assay was performed as described previously with minor modifications (Ren et al., 2022 (link)). Total RNA from the WT and the alkbh10b mutant were extracted and heated at 95 °C for 5 min to remove the secondary structure and then was chilled on ice. RNA was spotted onto Hybond™‐N+ membrane. The membrane was crosslinked using an HL‐2000 HybriLinker for 5 min and then washed with TBST (TBS with Tween‐20) for 5 min to remove the unbound RNA and blocked in 5% non‐fat milk for 1 h at room temperature. The membrane was then incubated with an m6A antibody (1:2000; ABclonal) overnight at 4 °C. The membrane was washed using TBST and incubated with the secondary antibody for 1 h at room temperature. Finally, the super ECL detection reagent (Yeasen) was used for visualization.
10
Western Blot Analysis of Acetic Acid Stress
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed as previously described with some modifications (47 (link)). Briefly, equal numbers of cells were collected when strains grew to the exponential phase in fermentation with acetic acid stress. Cells were treated with the following buffers: (i) distilled water, (ii) 2 M sodium hydroxide-8% mercaptoethanol, and (iii) TAP extraction buffer (4 mM HEPES-KOH [pH 7.5], 10% glycerol, 0.35 M NaCl, 0.1% Tween 20, 1× protease inhibitor cocktail, 1 mM PMSF, and 1× phosphatase inhibitor complex III [Sangon Biotech, China]). The sediment of cells was resuspended in 2× SDS loading buffer (0.1 M Tris-HCl [pH 6.8], 4% SDS, 0.2% bromophenol blue, 20% glycerol, and 2% mercaptoethanol) and boiled for 10 min. Protein samples were separated by 12% SDS-PAGE and transferred to Immobilon-P polyvinylidene difluoride (PVDF) membrane. The blots were probed with antibodies against specific proteins, followed by incubation with goat anti-mouse or goat anti-rabbit secondary antibodies. The antibodies used in this study are listed in Table S4. The specific proteins were visualized by super ECL detection reagent (Yeasen, China) in FluorChem FC3 (Proteinsimple, USA).
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!