The hippocampus was isolated and lysed in radio immunoprecipitation assay buffer (Beyotime, Shanghai, China) containing protease and phosphatase inhibitors. The protein concentration was measured using a bicinchoninic acid protein assay kit (Melone Pharmaceutical Co., Ltd., Dalian, China). An aliquot of 40 µg of total protein was separated by size via 10% sodium salt-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). After blocking with 5% non-fat milk (Beyotime) in Tris-buffered saline containing 0.1% Tween-20 (Beyotime) for 1 hour, the membranes were incubated overnight at 4°C with the following primary antibodies: anti-cofilin antibody (1:1000; rabbit; ab42824, Abcam, Cambridge, MA, USA), anti-cofilin (phospho S3) antibody (1:1000; rabbit, ab12866, Abcam), and anti-beta-actin antibody (1:1000; rabbit; ab8227, Abcam). The membranes were then incubated with horseradish peroxidase-labeled secondary antibodies (1:3000; goat anti-rabbit; A16104SAMPLE, Invitrogen, Waltham, MA, USA) for 1 hour at room temperature. The blots were visualized using the Chemistar High-sig enhanced chemiluminescence Western Blotting Substrate (Tanon, Shanghai, China). All protein bands were analyzed using ImageJ software (National Institute of Mental Health, Bethesda, MD, USA) (Kim et al., 2014).
Tween 20
Manufactured by Beyotime
Sourced in China, United States
Tween 20 is a non-ionic surfactant commonly used in laboratory applications. It is a polyoxyethylene sorbitan monolaurate, used to solubilize and stabilize proteins, enzymes, and other biomolecules. Tween 20 is a versatile reagent that can be used in various biochemical and analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Nonfat milk, by Beyotime (3 mentions)
Pbs buffer, by Beyotime (3 mentions)
Polyvinylidene fluoride (pvdf), by Beyotime (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Enhanced chemiluminescence, by Beyotime (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Ab42824, by Abcam (1 mentions)
Ab12866, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (1 mentions)
Tris hcl buffer, by Beyotime (1 mentions)
Protease and phosphatase inhibitor cocktail, by Beyotime (1 mentions)
Ab76420, by Abcam (1 mentions)
Piercetm ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp, by Cell Signaling Technology (1 mentions)
20 protocols using tween 20
1
Quantification of Cofilin and Phospho-Cofilin in Mouse Hippocampus
2
Immunohistochemical and Immunofluorescence Analysis of F4/80+ Macrophages in Bladder Tissues
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The paraffin-embedded bladder tissues were cut into 4 μm sections for immunological analysis. For immunohistochemical staining, the sections were deparaffinated, hydrated and incubated with primary F4/80 antibodies at 4 °C overnight. After washing in Tris-HCl buffer (Beyotime Biotechnology) mixed with 0.1% Tween 20 (Beyotime Biotechnology), these sections were continually incubated with a secondary antibody labeled with hydrogen peroxide oxidoreductase. All these stains were visualized with an Olympus microscope (Tokyo, Japan). For immunofluorescence staining, after the sections were incubated with the primary F4/80 antibody at 4 °C overnight and the secondary antibodies for 1 h at room temperature; appropriate second primary antibodies (CD86, with dilution 1:80/CD163, dilution 1:100) were applied at 4 °C overnight, and the second secondary antibodies were applied for another 1 h at room temperature. Then, the sections were counterstained with 4,6-diamino-2-phenylindole. Images were collected with a Zeiss Axio Scan.Z1 scanner (Carl Zeiss, Jena, Germany).
3
Adipocyte Protein Expression Analysis
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The adipocytes were lysed with RIPA Lysis Buffer (Cat. No. P0013 C, Beyotime Biotech. Shanghai, China) as instructed by protocol of manufacturer. The lysates were separated with 15% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE, Amresco Inc., Solon, OH, USA), and electro-transferred onto PVDF (Millipore, Boston, MA, USA). PVDF membranes were blocked with 5% defatted milk in PBS containing 0.05% Tween-20 (Beyotime Biotech, Co. Int, Beijing, China), with pH 7.5. Then, the PVDF membranes were incubated using rabbit anti-rat Bax polyclonal antibody (1: 2000; Cat. No. ab53154, Abcam Biotech., Cambridge, Massachusetts, USA), rabbit anti-rat caspase 3 polyclonal antibody (1: 2000; Ca. No. ab4051, Abcam Biotech.) and rabbit anti-rat GAPDH polyclonal antibody (1: 2000, Cat. No. ab9485, Abcam Biotech.) at 4 °C overnight. Subsequently, the PVDF membranes were treated using 1:3000 horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cat. No. AQ132P, Sigma-Aldrich, St. Louis, Missouri, USA). Western blot bands were visualized using BeyoECL Plus kit (Cat. No. P0018S, Beyotime Biotech. Shanghai, China). Eventually, western blot bands were imaged and analysed using LabworksTM Analysis Software (version: 4.6, Labworks, Upland, CA, USA).
4
Western Blot Analysis of Cellular Proteins
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With a phosphatase and protease inhibitor cocktail (Beyotime, China), cells were lysed and sonicated in RIPA (Beyotime, China) lysis solution. Proteins were separated by SDS‒PAGE and then transferred to a PVDF membrane (Merck Millipore, USA). Following 1 h at room temperature in a buffer containing 5% skim milk (BD Biosciences, USA) and 0.1% Tween 20 (Beyotime, China), the membranes were blocked. The membrane was then treated with the primary antibodies specified overnight at 4 °C, followed by incubation with goat anti-mouse or anti-rabbit secondary antibodies conjugated with horseradish peroxidase (HRP) (Cell Signaling Technology, USA) for 1 h. PierceTM ECL western blotting substrate (Thermo Fisher, USA) and Bio-Rad ChemiDoc Touch were used to detect and analyze the proteins. Antibodies against the following targets were used in this study: phospho-mTOR, mTOR, LC3B, P62, cleaved caspase 3, γ-H2A (5536, 2983, 3868, 23214, 9661, 2577, Cell Signaling Technology, USA), phospho-RPA2 (381220, Zen BioScience, China), actin (A5441, Sigma‒Aldrich, USA), RPA2 (ab76420, Abcam, USA), and Ki-67 (14-5698-82, Thermo Fisher, USA).
5
Quantitative Analysis of Nrf2 and Keap1
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Western blotting was performed as described in our previous study (Wang et al., 2019 (link)). Tween-20, RIPA buffer, protease inhibitor cocktail, bicinchoninic acid assay, 5 × loading buffer, SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels kit, and horseradish peroxidase-linked secondary antibodies were purchased from Beyotime Biotechnology (Shanghai, China). β-Actin was used as the loading control, and the protein bands of each sample were determined by the Alpha Imager 2200 software (Alpha Innotech Corporation, CA, United States). The antibody against β-actin was obtained from Bimake (Shanghai, China), and the antibodies against nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch like-ECH-associated protein 1 (Keap1) were purchased from Proteintech (Wuhan, China).
6
Aptamer-based PD-L1 Detection Protocol
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The Biotin-PD-L1 aptamer, Cy3-PD-L1 aptamer, Scambled-PD-L1 aptamer were all synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) The sequences are shown in Table S1 . Streptavidin-ALP made by Bioss Antibodies (catalog number: bs-0437p-AP). L-Argnine, Sodium Chloride, and Magnesium chloride made by Macklin Biochemical Co., Ltd. (Shanghai, China) Tween-20 made by Beyotime. To rotate the tubes we used 3D Rotary made by MIULAB. Water purified by Mili-Q portable benchtop system was used throughout all the experiments and buffers preparation.
7
Immunofluorescence Assay for Viral Infection
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Cells were infected with virus and simultaneously treated with the indicated concentrations of compounds for 24 h. The infected cells were fixed by 4% paraformaldehyde and permeabilized by 0.5% Triton X-100 followed by blocking in phosphate buffer saline (PBS; HyClone, Logan, Utah, USA) containing 1% bovine serum albumin (Applygen, Beijing, China) for 2 h at room temperature. Cells were then incubated with a double-stranded RNA (dsRNA) antibody (Cat# 10010200, 1:200; SCICONS, Szirák, Hungary) or HCoV-OC43 N protein antibody (Millipore, 1:200) overnight at 4 °C. After washing with PBS containing 0.1% (v/v) Tween 20 (Beyotime Institute of Biotechnology, Shanghai, China) for 3 times, the samples were reacted with Alexa Fluor-488 conjugated secondary antibody (Cat# HS231-01, 1:200; TransGen Biotech) for 1 h at room temperature away from light. Later, the nucleus was counterstained with Hoechst 33342 (Beyotime Institute of Biotechnology) for 30 min in the dark. Images were captured by a fluorescence microscope (Olympus IX71, Tokyo, Japan).
8
Quantifying SARS-CoV-2 RBD Antibodies by ELISA
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Binding titer: antigen-specific IgG antibody in plasma was detected by an indirect ELISA as previously described (23 (link)). Briefly, microplates (Corning, New York, USA) were coated with 1 μg/ml of wild-type RBD or various mutant RBDs (Sino, Shanghai, China) in 100 µl 0.05 M carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4°C. Plates were washed with PBS containing 0.5% Tween 20 (Beyotime, Shanghai, China) and blocked with PBS containing 5% (v/v) fetal bovine serum (FBS) (Hyclone, Utah, USA) at 37°C for 2 h. After washing three times, diluted plasmas (1:50) in PBS containing 0.5% (v/v) FBS were added and incubated for 2 h at 37°C. After three additional washes, 1:5,000 diluted goat anti-mouse IgG horseradish peroxidase conjugate (Proteintech, Chicago, USA) was added and further incubated for 2 h at 37°C. Plates were read at 450 nm on a microplate reader (Thermo scientific, Massachusetts, USA).
9
Immunofluorescence Staining of Testis Tissues
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Frozen testis sections and cultured cells were fixed with 4% paraformaldehyde for 10 min and washed twice with PBS. Later, they were blocked with 5% goat serum (Gibco) for at least 1 h and incubated with antibodies for 2 h (1:200 dilution in PBS). After being washed with PBST containing 0.1% Tween 20 (Beyotime), they were incubated with HRP-conjugated goat anti-rabbit IgG (Bioss) or HRP-conjugated goat anti-mouse IgG (Bioss) (1:2000 dilution in PBS) for 2 h. Antibody signals were colored using the TSA Plus Fluorescence Systems (PerkinElmer Life Science, Waltham, MA, USA). Nucleus was stained by propidium iodide. Photographs were imaged by a Zeiss SML800 laser scanning confocal microscope (Zeiss, Jena, Germany) or a Leica TCS SP5 laser scanning confocal microscope (Leica).
10
Western Blot Analysis of Protein Samples
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The samples were re-suspend using SDS-PAGE sample buffer (Beyotime Beotech. Shanghai, China), and then heated at 95°C for 10 minutes. The treated samples were loaded onto the 8% SDS-PAGE and separated using an electrophoresis apparatus (Bio-Rad Laboratories, Hercules, CA, USA). The separated samples were then transferred onto the PVDF membrane (Amresco Inc., Solon, OH, USA) on ice for 1 hour. PVDF membranes were blocked using 3% non-fat-dry milk diluting in PBS containing the 0.1% Tween 20 (Beyotime Biotech.). The PVDF membranes were incubated with relevant antibodies and following incubated using the horseradish peroxidase (HRP)-conjugated rabbit secondary antibodies. The western blotting signals were developed using SuperSignal™ west Dura Chemiluminescent Substrate Regent (Cat. No. #34076, Thermo Fisher Scientific) and then detected using the ChemiDoc™ (Bio-Rad Laboratories, Hercules, CA, USA).
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