Levels of SIRT2, FPN1, GPX4 and ACSL4 proteins were detected by western blotting. Protein lysates were prepared from the spinal cord (L4-6). Subsequently, 48 μg protein samples were separated using a 12% gradient sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) gel and transferred to polyvinylidene difluoride (pore size of 0.45 μm) membrane (Bio-Rad). The membranes were blocked in non-fatty dry milk for 2 h at 37°C and then incubated with primary antibodies, including anti-SIRT2 (1:500, ab51023, Abcam), anti-FPN1 (1:1,000, NBP1-21502, Novus), anti-NRF2 (1:1,000, ab137550, Abcam), anti-GPX4 (1:5,000, ab125066, Abcam), anti-ACSL4 (1:10,000, ab155282, Abcam), and anti-GAPDH (1:10,000, 60004-1-Ig, Proteintech) antibodies at 4°C overnight, and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. We analyzed the density of protein bands using the NIHA ImageJ software.
Polyvinylidene difluoride
Manufactured by Bio-Rad
Sourced in United States
Polyvinylidene difluoride (PVDF) is a type of laboratory equipment used in various applications. It is a synthetic polymer material with unique properties that make it suitable for use in various scientific and industrial settings. PVDF is a durable, chemically resistant, and thermally stable material, making it suitable for a range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Nitrocellulose, by Bio-Rad (4 mentions)
Odyssey blocking buffer, by LI COR (3 mentions)
Non fat milk, by Bio-Rad (2 mentions)
Tris glycine sodium dodecyl sulfate polyacrylamide gels, by Bio-Rad (2 mentions)
Ecl kit, by Cytiva (2 mentions)
Odyssey clx infrared imaging system, by LI COR (2 mentions)
Nupage bis tris polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Ab51023, by Abcam (1 mentions)
Ab155282, by Abcam (1 mentions)
Ab125066, by Abcam (1 mentions)
Nbp1 21502, by Novus Biologicals (1 mentions)
Ab137550, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Bio-Rad (1 mentions)
34 protocols using polyvinylidene difluoride
1
Evaluation of Oxidative Stress Markers
2
Western Blot Immunodetection Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblotting was performed as described previously14 (link). Briefly, cell lysates were made in lysis buffer (pH 7.5) containing 1 mM EDTA, 50 mM Tris, 150 mM NaCl, 0.5% Triton X-100, 0.5% NP-40, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 5 mM sodium vanadate, and 1 μg/ml leupeptin, aprotinin, and pepstatin. Proteins were then separated by SDS-PAGE gels and further transferred to the membranes of polyvinylidene difluoride (Bio-Rad, Hercules, CA, USA). After blocking the membranes with 5% nonfat milk for 1 h at room temperature, they were incubated with primary antibodies as indicated. The membranes were then washed and incubated at room temperature with peroxidase-conjugated secondary antibodies for 1 h. After 5 times of washing, immunodetected bands on the membranes were visualized by taking chemiluminescent images on X-ray films with the enhanced chemiluminescence (ECL) system (PerkinElmer, Waltham, MA, USA) as per the manufacturer’s instructions. All uncropped scans are shown in Supplementary Fig. 13 for the western blots.
3
Exosomal Protein Isolation and Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated exosomes were lysed using a RIPA buffer (Sigma-Aldrich Co.) containing 50 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 1.0% Igepal, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate and the protease inhibitor cocktail. The total protein concentration in exosomes extracts was determined by the bicinchoninic acid assay (Thermo Scientific, Waltham, MA). Subsequently, exosomes were resuspended in the loading buffer and boiled at 99 °C for 5 min. Equal volume or equal protein amount of sample was mixed with reducing Laemmli-buffer and was loaded on 4–20% Tris-glycine sodium dodecyl sulfate-polyacrylamide gels (Bio-Rad, Hercules, CA, US), and electrophoresed. Proteins were transferred to polyvinylidene difluoride (Bio-Rad, Hercules, CA, US). Membranes were blocked in 5% non-fat milk (Bio-Rad, Hercules, CA, US) in Tris-buffered saline supplemented with 0.05% Tween-20 (TBS-T) for 2 h, and then were incubated with primary antibodies: anti-CD63 [1:500; Santa Cruz Biotechnology, Dallas, TX, US sc:15363], anti-Histon H3 [1:500; St John’s Labolatory STJ93527] for 16 h at 4 °C. After 3 washes in TBS-T, membranes were incubated with corresponding HRP-conjugated secondary antibodies for 2 h at room temperature and washed in TBS-T. Signals were visualized after incubation with enhanced chemiluminescence kit (Bio-Rad, Hercules, CA, US) by Chemidoc Touch (Bio-Rad, Hercules, CA, US).
4
Analyzing Protein Expression in SW-480 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SW-480 cells were plated in 6-well plates at approximately 1 × 106 cells/well and media were replaced with 10% FBS/RPMI. After 24 h incubation, the cells were treated with different concentrations of LER for 24 h. The harvested cells were washed with PBS twice and then lysed with cell lysis buffer (150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl, pH 7.5, 2 mM storage) for 30 min at 4°C. The homogenates were centrifuged at 13,000 × g for 10 min to isolate the supernatants. Protein concentrations were determined using the pierce based on bicinchoninic acid protein assay. Thirty grams of the protein from the supernatants were then separated on 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride (Bio-Rad) membranes. After transfer, the membrane was blocked at room temperature for 1 h with 5% BSA in Tris-buffered saline-Tween (TBST; 20 mM Tris, 500 mM NaCl, pH 7.5, 0.1% Tween 20). Then, the membranes were incubated with primary antibodies for 1 h, after three washes in TBST for 5 min, followed by incubation with secondary antibody for 1 h at room temperature. The immunoreactive protein bands were detected by using an enhanced chemiluminescence detection system. Blots were developed with a Mini BIS Image Analysis System (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel).
5
Immunoprecipitation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We performed immunoprecipitation as described previously [72 (link)]. In brief, total cell lysates were collected and then were immunoprecipitated by incubation with antibodies against Flag (1:100, Cell Signaling Technology, Boston, MA, USA) and HA (1:100, Cell Signaling Technology). SDS–PAGE was used to separate immunoprecipitates and then was blotted onto a polyvinylidene difluoride (BioRad Laboratories, Hercules, CA, USA) membrane. The membrane was incubated with antibodies against HA (1:2000, Cell Signaling Technology) and Flag (1:2000, Cell Signaling Technology) respectively and visualized by enhanced chemiluminescence (ECL Kit; Amersham Biosciences, Slough, Buckinghamshire, UK). Whole-cell lysates were separated by SDS–PAGE and blotted on polyvinylidene difluoride membranes (Millipore) for western blotting analysis reprobed with appropriate peroxidase-conjugated HA and Flag antibodies. Blots were visualized by enhanced chemiluminescence (ECL Kit; Amersham Biosciences).
6
Western Blot Analysis of Neural Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein lysates were generally extracted from cells using a protein extraction buffer (Novagen, Madison, WI, USA) containing protease inhibitor (Calbiochem, San Diego, CA, USA). After 30 min incubation on ice, protein lysates were clarified by centrifugation at 12,000 g, 10 min. Total protein concentration was routinely determined using protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Total proteins (30 μg) per sample were separated by 10% SDS–polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride (Bio-Rad Laboratories) membrane. After blocked with 5% nonfat milk for 1 h, specific primary antibodies were incubated overnight at 4°C, and then secondary antibodies were further incubated for 1 h. Equal protein sample were verified by β-actin. Chemiluminescent detection was performed using a chemiluminescence detection kit (AbFrontier, South Korea) by the ChemiDoc Touch imaging system (Bio-Rad Laboratories). The primary antibodies used in this study including antinestin antibody (1:1,000), anti-MAP2 antibody (1:1,000), anti-GFAP antibody (1:1,000), anti–p-GSK-3β(Ser9) antibody (1:1,000), anti–GSK-3β antibody (1:1,000), anti–non-p-β-catenin (active) antibody (1:1,000), anti–β-catenin antibody (1:1,000), and anti–β-actin antibody (1:5,000).
7
Protein Expression Analysis of EMT Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Frozen tissues were lysed in the lysis buffer, and the BCA protein assay kit (Beyotime, Pudong, Shanghai, China) assisted in measuring the concentration of protein. 12% SDS-PAGE gel was used to separate proteins which were then transferred to the PVDF membranes (polyvinylidene difluoride; Bio-Rad). After 2 h of blocking with 5% skim milk, the membrane received 2 h of incubation with primary antibodies and 1 h of incubation with the secondary antibody at room temperature. ImageJ software (National Institutes of Health, Bethesda, MD, USA) helped to analyze the western blot results. Primary antibodies of E-cadherin (ab241677, Epitomics Inc., Burlingame, CA, USA), Vimentin (ab45939, Epitomics Inc., Burlingame), N-cadherin (ab76011, Epitomics Inc., Burlingame), GAPDH (ab8245, Abcam Inc.), and BSG (ab188190, Epitomics Inc., Burlingame) were used in the immunoblotting assays.
8
Western Blot Protein Separation and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Before cell lysis, the cultured cells were washed with phosphate-buffered saline (PBS) once. Then, cells were directly lysed with 1 × Laemmli buffer (2% sodium dodecyl sulfate [SDS], 5 mM DTT, 10% glycerol, 0.002% bromophenol blue, and 63 mM Tris-HCl at pH = 6.8) and boiled at 98° C for 5 min. The proteins with different sizes were then separated by SDS-PAGE using 6–15% gels. The separated proteins were then transferred onto polyvinylidene difluoride (1620177, Bio-Rad) membranes. The membranes were briefly activated in methanol and blocked with 5% milk in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature, followed by incubation of target protein-specific antibodies at 4 °C overnight. Then, the membranes were washed three times for 10 min with TBST and hybridized with horseradish peroxidase-conjugated secondary antibodies (in TBST containing 5% milk) at room temperature for another 1 h. After washing three times (5 min) in TBST, the bands on the membranes were visualized by adding the enhanced chemiluminescence substrate (RPN2235, GE Healthcare). All the images were captured under the ChemiDoc gel imaging system (Bio-Rad), and densitometry analysis was performed by Image Lab software (Bio-Rad). The internal controls were applied to normalize the target protein, and the means of quantifications was shown in graphs.
9
Protein Extraction and Quantification Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Culture media were collected, treated with 1 mM EDTA, and centrifuged at 2500 × g at 4 °C for 1 min to settle cellular debris. Protein content of cell culture media samples was precipitated with the methanol–chloroform method, as described previously [18 (link)]. Cells were lysed in ice-cold RIPA buffer (20 mM Tris-HCl pH = 7.4, 150 mM NaCl, 1% v/v Triton X-100, 1% m/v Na-deoxycholate, 1 mM Na3VO4, 10 mM NaF, 0.1% m/V SDS, 1 mM EDTA and 1 mM Pefabloc), centrifuged at 9500 × g at 4 °C for 30 min. Protein concentration was determined with the bicinchoninic acid method (Santa Cruz Biotechnology, Dallas, TX, USA). Proteins were electrophoresed with standard denaturing SDS-PAGE procedures and blotted on polyvinylidene difluoride (Bio-Rad, Hercules, CA, USA) membranes. After blocking, the membranes were incubated with primary antibodies overnight, washed repeatedly, and incubated with the secondary antibodies. Antibodies used are shown in Table 1 . The immunoreaction was visualized with the Bio-Rad Clarity Chemiluminescent Substrate in a ChemiDoc MP imaging system (Bio-Rad). Quantifications were performed using the ImageLab software version 5.2 (Bio-Rad).
10
Subcellular Protein Fractionation of Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CV1-derived and HapI-derived cells were infected as indicated and then fractionated into soluble cytoplasmic (Cyto.), membrane (Memb.), nuclear (Nuc.), chromatin-bound (Chrom.) and cytoskeletal (Cytoskel.) fractions using a subcellular protein fractionation kit for cultured cells (Thermo Scientific, catalog no. 78840) according to the manufacturer’s instructions with the addition of phosphatase inhibitors. Lamin A/C was used as a nuclear protein control that is present in soluble fractions and fractions bound to the chromatin and cytoskeleton, along with H2B that localizes specifically to the chromatin nuclear fraction. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a predominant cytosolic and minor membrane-associated protein control. Sample lysates were resolved by SDS-PAGE, transferred to polyvinylidene difluoride (Bio-Rad), and developed as described above.
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!