The appropriate amount of peritoneal tissue should be taken for total protein extraction, and the concentration of total protein extracted should be determined by the BCA protein method. The same amount of protein (20 μg) is removed and added to 8%–12% SDS-PAGE gel for separation of proteins and transferred to polyvinyl fluoride membrane (Millipore). Dilutions of the following antibodies were made and cocultured with the membrane overnight at 4°C: E-cadherin (1:1,000), Fibronectin (1:2000),N-cadherin (1:2,000), α-SMA (1:1,000), vimentin (1:1,000), phospho-Smad2 (1:1,000), phospho-Smad3 (1:2,000), TGF-β1 (1:1,000), phospho-NF-κB p65 (1:1,000), NOX2 (1:5000), NOX4 (1:1000), phospho-Erk1/2 (1:1000) and GAPDH (1:5000). Then incubate the membrane with horseradish peroxidase bound secondary antibody (1:5000) for 1 h at room temperature. The ECL system and Bio Rad electrophoretic image analyzer were used to observe the immune reaction zone after the developer was added.
Polyvinyl fluoride membrane
Manufactured by Merck Group
Sourced in Germany, United States
Polyvinyl fluoride membrane is a type of lab equipment used for filtration and separation processes. It is a durable, chemically resistant material that can be used to filter a variety of liquids and gases. The membrane's core function is to act as a physical barrier, allowing the passage of certain components while retaining others.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ecl system, by Bio-Rad (1 mentions)
Image station 2000 mm, by Kodak (1 mentions)
Luminata forte, by Merck Group (1 mentions)
Ab214666, by Abcam (1 mentions)
Ab6789, by Abcam (1 mentions)
P0018fs, by Beyotime (1 mentions)
Ab8245, by Abcam (1 mentions)
P0012, by Beyotime (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Skim milk, by BD (1 mentions)
Goat anti rabbit hrp, by Beyotime (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Fd8030, by Fudebio (1 mentions)
Fdm007, by Fudebio (1 mentions)
Anti s1pr2, by Proteintech (1 mentions)
Anti akt, by Wanlei (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
9 protocols using polyvinyl fluoride membrane
1
Evaluating Epithelial-Mesenchymal Transition
2
Quantitative Western Ligand Blotting of Serum IGFBPs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative Western ligand blotting of serum IGFBP2, IGFBP3 and IGFBP4 were performed as previously described [13 (link)] on samples obtained on day 0, 14 and 18. To perform the Western ligand blot, serum samples were diluted with sample buffer (Lämmli buffer/bromophenol blue, EDTA) and boiled (5 min). Electrophoresis (Twin ExW S Perfect Blue; Peqlab Biotechnologie, Germany) was then performed on 11% SDS polyacrylamide gel. The separated proteins were transferred to a polyvinyl fluoride membrane (Millipore, Germany), and these blots (Semi Dry-Blotter Perfect Blue; Peqlab Biotechnologie) were incubated with blocking buffer and biotin-labelled IGF2 followed by washing steps and incubation with blocking buffer and streptavidin peroxidase-conjugate (IBT, Germany). After incubation with a detection reagent (Luminata Forte; Millipore), chemiluminescence was detected (Kodak Image Station 2000MM; Kodak, USA) and evaluated (GelAnalyzer 2010a by Dr. Istvan Lazar; GraphPad 4.0; GraphPad Software, USA). The intra- and inter-assay CV was 9.9 and 15.5% for IGFBP2, 7.8 and 19.4% for IGFBP3 and 7.7 and 18.1% for IGFBP4, respectively. The lowest detection limit was 0.2 ng/µL for IGFBP2, 1.1 ng/µL for IGFBP3 and 0.3 ng/µL for IGFBP4.
3
Western Blot Quantification of KLF4 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total proteins of the cells were isolated by a radioimmunoprecipitation assay buffer (P0013E, Beyotime, Shanghai, China) and then quantified by a bicinchoninic acid protein assay kit (P0012, Beyotime, Shanghai, China). After the loading buffer was added, the proteins were denatured in boiling water. The protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinyl fluoride membrane (Millipore, Bedford, MA, United States). After that, the membrane was blocked in 5% skim milk for 30 min at room temperature. Next, the proteins were incubated with primary antibodies anti-KLF4 (ab214666, Abcam, Cambridge, United Kingdom) and anti-GAPDH (ab8245, Abcam, Cambridge, United Kingdom) overnight at 4°C and then incubated with the secondary antibody (ab6789, Abcam, Cambridge, United Kingdom) at room temperature for 1 h. Lastly, the protein bands were visualized using ECL chemiluminescence solution (P0018FS, Beyotime, Shanghai, China), and then, the gray value of the bands was quantified by Image LabTM Software.
4
Exosomal Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To confirm the success of EX isolation, ~ 30–40 μg of exosomal protein was loaded onto a polyacrylamide gel (5% stacking and 10% resolving gel). After running the gel at 90 V for 20 min, followed by 120 V for 60 min in running buffer, the proteins were transferred to a polyvinyl fluoride membrane (0.45 μm; Millipore) and blocked with 5% skim milk (BD, Lincoln, NE, USA) for 2 h. The membranes were incubated overnight with primary antibodies (Abs) against CD63, CD9, C81 (rabbit pAb; SBI, Palo Alto, CA, USA), pregnancy-specific globin (PSG1), nestin (NES), progesterone receptor [PGR] (rabbit pAb; Absin, Shanghai, China), L1 cell adhesion molecule [L1CAM] (rabbit pAb; Abcam, Cambridge, UK), and placental alkaline phosphatase [PLAP](mouse mAb; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at 4 °C. The Ab dilution ranged from 500- to 1000-fold times, as appropriate. The next day, the membranes were washed and incubated with secondary antibodies (goat anti-rabbit-HRP; Beyotime, Shanghai, China). The blotted proteins were visualized using an enhanced chemiluminescence kit (Pierce Biotechnology, Rockford, IL, USA), and the signal was detected using Saga (Sage Sciences, Lincoln, NE, USA).
5
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 obtained by lysing the transfected FaDu cells. A protein bicinchoninic acid assay kit (Beyotime Institute of Biotechnology, Shanghai, China) was used to measure the protein concentration. Equal amounts of protein were loaded onto the SDS-PAGE and then transferred to a polyvinyl fluoride membrane (EMD Millipore, Darmstadt, Germany). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies against PD-L1, E-cadherin, N-cadherin, Vimentin, Akt, p-Akt, p-mTOR and β-actin overnight at 4°C. Further, the membranes were incubated with appropriate secondary antibodies in the next day. The immunoreactive bands were detected using the enhanced chemiluminescence reagent (EMD Millipore, Darmstadt, Germany) and the gray values of protein bands were evaluated by Image J software (version 1.80; National Institutes of Health, Bethesda, MA, USA).
6
Western Blot Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tissues and cells were decomposed with radioimmunoprecipitation assay buffer (Beyotime), separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to a polyvinyl fluoride membrane (EMD Millipore). Transition to primary antibody and primary antibody reducing solution (Thermo Fisher Scientific). After washing with Tris‐buffered saline with Tween 20 (TBST), the membrane and primary antibody were separated at room temperature for 1 h, and then washed with TBST three times.
7
Western Blot Analysis of Protein Targets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted using RIPA buffer (Beyotime, Shanghai, China), and the concentration was determined by a BCA kit (ThermoFisher Scientific). An equivalent amount of proteins was isolated by SDS-PAGE, and transferred to polyvinyl fluoride membrane (Merck KGaA). After incubation with primary antibodies overnight at 4°C, and incubation with horseradish peroxidase-conjugated secondary antibodies (FDM007 and FDR007, Fudebio, Hangzhou, China) for 2 h. The membranes were treated with the enhanced chemiluminescent reagents (MILLIPORE, WBKLS0500). The signals were examined by ChemiDox (bio-rad, USA) with the treatment of an enhanced chemiluminescence kit (FD8030, Fudebio, Hangzhou, China). The primary antibodies involved in the present study were GAPDH (1:1000, Abcam), anti-S1PR2 (1:500, Proteintech), anti-AKT (1:1000, Wanleibio), anti-p-AKT (Ser473) (1:1000, Wanleibio).
8
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein samples used for western blot were extracted with RIPA lysis buffer (Beyotime Biotechnology, China) supplemented with protease inhibitors (Roche, China). Protein concentrations were determined with the bicinchoninic acid (BCA) assay kit (Thermo scientific, USA). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis, transferred onto polyvinyl fluoride membranes (Merck KGaA, Darmstadt, Germany), and incubated with primary (rabbit anti-Thy-1 (ab225, Abcam, US), mouse anti- integrin β3 (sc-46655, Santa Cruz, USA), rabbit anti-LC3 I/II (L7453, Sigma, USA), rabbit anti-p62/SQSTM1 (#5114, Cell Signaling Technology, USA), rabbit anti-Akt (#4691, Cell Signaling Technology, USA), rabbit anti-p-Akt (#4060, Cell Signaling Technology, USA), rabbit anti-mTOR (#2983, Cell Signaling Technology, USA), rabbit anti-p-mTOR (#2971, Cell Signaling Technology, USA) and mouse anti-GAPDH (30201ES20, Yeasen, China) antibodies, respectively. This was followed by incubation with the appropriate secondary antibodies, including goat anti-mouse (A0216, Beyotime, China) and goat anti-rabbit (A0208, Beyotime, China) antibodies, respectively. Signals were detected using the ECL Plus Western blotting system kit (Beyotime Biotechnology, China); band intensity was measured with the Image LabTM software (Bio-Rad, USA).
9
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Equal amount of protein extracts were lysed using radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich; Merck KGaA). Cells were centrifuged in a microcentrifuge at 12,000 × g for 15 min at 4°C to collect the supernatant. Protein concentration was determined using a Bicinchoninic Acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). A total of 30 µg protein were separated by 10% SDS-PAGE and then transferred onto polyvinyl fluoride membranes (Merck KGaA). The membranes were blocked with 5% fat-free milk in Tris-buffered saline for 1 h, and incubated with anti-PTPRO (1:1,000; catalog no. sc-365354; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-β-actin (1:2,000; catalog no. 4970, Cell Signaling Technology, Inc., Danvers, MA, USA) primary antibodies at 4°C overnight. The membranes were washed three times with Tris-buffered saline containing 0.1% Tween and incubated for 2 h at room temperature with a horseradish peroxidase-conjugated goat anti-rabbit (catalog no. 7074; 1:1000; Cell Signaling Technology, Inc.) or anti-mouse secondary antibody (catalog no. sc-516102, 1:2000, Santa Cruz Biotechnology). Proteins were visualized using a Bio-Rad ChemiDoc Imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, 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!