The collected Raw264.7 cells and bone surrounding soft tissues were homogenized and lysed in lysis buffer including RIPA and phenylmethanesulfonyl fluoride (PMSF; Beyotime Institute of Biotechnology, Haimen, Jiangsu, China). The lysates (30 μg/lane) were separated on 10% gels and were transferred onto polyvinylidene difluoride membranes (Beyotime Institute of Biotechnology). The membranes were blocked with 5% (w/v) BSA in Tris-buffered saline with 0.1% (w/v) Tween 20 (TBST) and probed with primary antibodies: mouse anti-ABCA1 (1: 1000; Abcam Biotechnology), rabbit anti-ABCG1 (1: 2500; Abcam Biotechnology), rabbit anti-Cav-1 (1: 1500; Abcam Biotechnology), rabbit anti-TNF-α (1: 1000; Abcam Biotechnology), rabbit anti-IL-6 (1: 1000; Cell Signaling Technology), rabbit anti-IL-1β (1: 1,000; Abcam Biotechnology), and mouse anti-β-actin (1: 4000; Proteintech, Rosemont, USA) at 4°C overnight. After washing, the bound antibodies were detected using optimal horseradish peroxidase-conjugated secondary antibodies: goat anti-rabbit IgG (1: 10 000) and goat anti-mouse IgG (1: 10 000; Merck Millipore, Darmstadt, Germany) and were visualized using the enhanced chemiluminescence reagents (Bio-Rad Laboratories, Hercules, USA). The relative levels of the target proteins to that of control β-actin were analyzed densitometrically using the Quantity One software (version 4.0, Bio-Rad).
Polyvinylidene difluoride membrane
Manufactured by Beyotime
Sourced in China, United States
Polyvinylidene difluoride (PVDF) membranes are a type of laboratory equipment used for various applications. PVDF membranes are known for their chemical resistance, thermal stability, and low protein binding properties. They are commonly used in techniques such as Western blotting, dot blotting, and protein transfer.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (8 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Ab181602, by Abcam (4 mentions)
Sds page, by Beyotime (4 mentions)
Ab205718, by Abcam (3 mentions)
Bca protein assay kit, by Applygen (2 mentions)
Horseradish peroxidase conjugated goat anti mouse or goat anti rabbit igg secondary antibodies, by ZSGB-BIO (2 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (2 mentions)
Ab6721, by Abcam (2 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (2 mentions)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Beyotime (2 mentions)
Bca kit, by Beyotime (2 mentions)
Bicinchoninic acid assay kit, by Beyotime (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Ecl kit, by Beyotime (2 mentions)
Enhanced chemiluminescence reagent, by Beyotime (2 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Mouse anti β actin, by Proteintech (1 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (1 mentions)
Goat anti mouse igg, by Merck Group (1 mentions)
41 protocols using polyvinylidene difluoride membrane
1
Western Blot Analysis of Inflammatory Markers
2
Protein Expression Analysis in Renal Cells
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Total proteins were isolated from the renal tissues and 786-O cells by using RIPA buffer, and protein concentrations were quantified using BCA Protein Assay Kit (Beyotime, Shanghai, China). Protein samples (35 μg/lane) were resolved by performing sodium dodecyl sulphate-polyacrylamide gel electrophoresis and were transferred onto polyvinylidene difluoride membranes (Beyotime). The membranes were incubated overnight at 4 °C with mouse anti-human FGL2 antibody (Ab) (dilution, 1:400; Abnova, Taiwan), rabbit anti-human GAPDH antibody (Ab) (dilution, 1:1000; Abcam, Cambridge, MA, USA), rabbit anti-human ERK1/2 antibody, mouse anti-human phospho-ERK1/2 antibody, rabbit anti-human p38 antibody or phospho-p38 antibody (all dilution, 1:1000; Beyotime, Shanghai, China) followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG secondary Abs (dilution, 1:3000; ZSGB-BIO, Beijing, China). Immunoblots were visualized using a ECL Western Blotting Detection System (Millipore, Billerica, MA, USA). GAPDH was used as a loading control.
3
ORF142 Protein Detection in LSDV-infected Cells
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MDBK cells infected or not infected with LSDV-WT were washed three times with cold PBS at specific time points and then harvested with lysis buffer containing 1% protease inhibitors (Beyotime). The protein concentration was determined with a BCA Protein Assay Kit (Beyotime). The cell extracts were resolved with sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Beyotime). Total ORF142 protein was detected with a rabbit anti-ORF142 antibody (stored in our laboratory), with rabbit anti-β-actin (Proteintech, Wuhan, China) as the loading control.
4
Investigating CD44-mediated Leukemia Cell Signaling
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The NB4 cell line was obtained from Meisen (Zhejiang, People's Republic of China). Fetal bovine serum and RPMI 1640 medium were purchased from HyClone (Logan, UT). Hyaluronan and human fibrinogen were obtained from Suolaibao (Beijing, People's Republic of China). Anti–phosphatidylinositol 3-kinase (PI3K) antibodies and anti–protein kinase B (Akt) antibodies were purchased from Wanleibio (Shanghai, People's Republic of China). Anti-mTor, anti–P-mTor, and anti-CD44 antibodies were purchased from Proteintech (Hubei, People's Republic of China). Human thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). The human CD44 enzyme-linked immunosorbent assay (ELISA) kit was obtained from Abcam (Stamford, CT). ATRA, ATO, and DNR were purchased from Sigma (St. Louis, MO). Polyvinylidene difluoride membranes, anti-mouse antibodies, and anti-rabbit antibodies were obtained from Beyotime (Shanghai, People's Republic of China). We prepared Alexa Fluor-488– or Alexa Fluor-647–conjugated lactadherin. Anti-CD41a and anti–P-selectin antibodies were purchased from Abcam.
5
Western Blot Analysis of Sperm Proteins
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Sperm samples were homogenized in 80 μl of an ice-cold lysis buffer containing a cocktail of protease inhibitors and lysed by sonication several times. These homogenates were centrifuged at 12,000×g for 15 min at 4 °C, and the supernatant was collected for the quantification of total proteins. Samples with equal protein quantity (50 μg/lane) were electrophoresed on a 10% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Beyotime, China). The membranes were blocked with 5% milk for 1 h at room temperature and then incubated with primary antibodies overnight at 4 °C. After washing with Tris-buffered saline with Tween 20, the membranes were incubated with secondary antibodies for 1 h at room temperature. Antibodies are listed in Supplementary Table 1 . The band intensities were quantified by densitometry using ImageJ analysis software (Research Services Branch).
6
Western Blot Analysis of EMT Markers
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Total protein was extracted using RIPA lysis buffer (Applygen, Beijing, China) containing phenylmethanesulfonyl fluoride (Beyotime), followed by quantitation with the BCA protein assay kit (Applygen). Then, protein (25 μg) from each sample was resolved by SDS-PAGE, followed by being transferred to polyvinylidene difluoride membranes (Beyotime). The membranes were incubated for 12 h at 4 °C by primary antibody against cleaved-caspase3 (cleaved-casp3) (1:500, ab2302; Abcam, Cambridge, UK), E-cadherin (1:800, ab15148; Abcam), N-cadherin (1:800, ab18203; Abcam), TGM2 (1:2000, ab137378; Abcam), and GAPDH (1:3000, ab70699; Abcam) after blocking with 5% non-fat dried milk. After that, the secondary antibody (1:5000, D110058; Sangon Biotech) was used to incubate the membranes for 2 h. Finally, the ECL kit (Applygen) was employed to visualize the protein bands. Protein levels were normalized to GAPDH and evaluated using ImageJ software.
7
Renal Protein Extraction and Western Blot Analysis
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Total proteins were isolated from the renal tissues using the RIPA buffer, and protein concentrations were quantified using the BCA protein assay kit (Beyotime, Shanghai, China). Protein samples (35 μg/lane) were resolved via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto polyvinylidene difluoride membranes (Beyotime, Shanghai, China). The membranes were incubated overnight at 4° C with rabbit anti-mouse fibronectin (1:1000), mouse anti-mouse α-SMA (1:200), mouse anti-mouse E-cadherin (1:500), rabbit anti-mouse Sphk1 (1:1000) (All from Abcam, Cambridge, MA, USA) followed by incubation with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG secondary antibodies (1:3000; ZSGB-BIO, Beijing, China). Immunoblots were visualized using the ECL Western blot Detection System (Millipore, Billerica, MA, USA). GAPDH was used as the loading control.
8
Expression and Purification of Pneumococcal Proteins
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Beijing Tsingke Biotechnology Co. (Ltd.) was entrusted to synthesize the target gene fragment, and the gene sequence was referred to as S. pneumoniae standard strain R6 (NC_003098.1). The recombinant expression pET28a plasmid that contained the in frame of the nanT1-tufT1-plyD4 DNA construct was identified by Sanger sequencing after transforming the E. coli DH5α competent strain. The target protein was expressed in E. coli BL21(DE3). Liquid cultures were induced overnight at 18°C with 0.4 mM isopropyl β-D-thiogalactoside (IPTG), lysed with Bugbuster HT (Novagen), and subsequently purified over a His-Select Ni2+-NTA gravity column (Cytiva, USA), anion exchange chromatography column (Cytiva, USA), gel filtration chromatography column (Cytiva, USA), and polymyxin B resin gravity column (GenScript, USA).
The purity of the fusion protein was evaluated by SDA-PAGE, while the concentration was determined by the bicinchoninic acid (BCA) method. For Western blot analysis, the purified fusion protein was subjected to 10% SDS−PAGE, electrophoretically transferred to polyvinylidene difluoride membranes (Beyotime Biotechnology, Shanghai, China) and detected by an indirect antibody immunoassay.
The purity of the fusion protein was evaluated by SDA-PAGE, while the concentration was determined by the bicinchoninic acid (BCA) method. For Western blot analysis, the purified fusion protein was subjected to 10% SDS−PAGE, electrophoretically transferred to polyvinylidene difluoride membranes (Beyotime Biotechnology, Shanghai, China) and detected by an indirect antibody immunoassay.
9
Western Blot Analysis of HOXC8 Protein
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Total protein was extracted using cell lysis buffer (Beyotime). Protein concentrations were determined using a bicinchoninic acid protein assay kit (Beyotime), and 50-µg samples were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Beyotime). The membranes were blocked in 5% bovine serum albumin, followed by incubation overnight with primary antibodies (anti-HOXC8: ab86236; anti-GAPDH: ab181602; Abcam, Cambridge, MA, USA) at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (ab6721, Abcam) for 2 hours at room temperature and the protein signals were visualized using a BeyoECL Plus kit (Beyotime) and observed using a ChemiDoc MP system (Bio-Rad, Hercules, CA, USA). HOXC8 levels were analyzed using Image J 1.44 software (NIH, Bethesda, MA, USA) with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a loading control.
10
Western Blot Analysis of IGF-1R Expression
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RIP assay lysis buffer (Beyotime Biotechnology Ltd.) was used to extract total protein. Protein quantification was performed using the Bradford Protein Assay Kit (Beyotime Biotechnology Ltd.,). Equivalent amounts of protein were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were transferred onto polyvinylidene difluoride membranes (Beyotime Biotechnology Ltd.) and blocked with 5% skimmed milk. The membranes were then incubated with primary antibodies against IGF-1R (ab182408; 1:1000 dilution; Abcam, Cambridge, MA, USA) or GAPDH (ab181602; 1:1000 dilution; Abcam) overnight at 4 °C. The membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (ab205718; 1:5000 dilution; Abcam) followed by detection with the BeyoECL moon protein detection kit (Beyotime Biotechnology Ltd.). GAPDH was used as a loading control.
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