DG75 cells (2 × 106) or exosomes (10 µg) were separated by SDS-PAGE (10%) and transferred to polyvinylidene difluoride membranes (Millipore). A total of 1 × 106 negatively selected B cells was incubated (37°C, 5% CO2) for 15 h with 100 µg BJABex or LCL1ex in 500 µl complete medium (48-well plate; Becton Dickinson). B cells were washed three times with PBS to remove unbound exosomes and incubated for the remaining 24 or 48 h in complete medium (37°C, 5% CO2). Cell lysates were separated by SDS-PAGE (NuPAGE 4–12% Bis-Tris Gel; Life Technologies) and transferred to polyvinylidene difluoride membranes (Millipore). Membranes were stained according to the manufacturer’s instructions with Abs against LMP1 (CS. 1.4; Dako), EBNA2 (PE2; Novocastra), HLA-DR (TAL.1B5; Dako), CD81 (H-121; Santa Cruz Biotechnology), and β-actin (I-19; Santa Cruz Biotechnology). Membranes were visualized with ECL Prime Western Blotting Detection Reagent (GE Healthcare) and exposed on CL-XPosure films (Nordic Biolabs).
Polyvinylidene difluoride membrane
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Polyvinylidene difluoride (PVDF) membranes are a type of lab equipment used for various applications. PVDF membranes are known for their chemical resistance, thermal stability, and mechanical strength. They are commonly used in filtration, separation, and transfer processes in laboratory settings.
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Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (225 mentions)
Ripa lysis buffer, by Beyotime (207 mentions)
Bca protein assay kit, by Beyotime (182 mentions)
Ripa buffer, by Beyotime (138 mentions)
Protease inhibitor cocktail, by Roche (137 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (123 mentions)
Ripa buffer, by Thermo Fisher Scientific (94 mentions)
Protease inhibitor cocktail, by Merck Group (92 mentions)
β actin, by Cell Signaling Technology (91 mentions)
Quantity one software, by Bio-Rad (88 mentions)
β actin, by Santa Cruz Biotechnology (87 mentions)
Gapdh, by Cell Signaling Technology (78 mentions)
β actin, by Merck Group (77 mentions)
Image lab software, by Bio-Rad (71 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (66 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (61 mentions)
β actin, by Abcam (59 mentions)
Gapdh, by Abcam (58 mentions)
Odyssey infrared imaging system, by LI COR (57 mentions)
Gapdh, by Santa Cruz Biotechnology (55 mentions)
4 719 protocols using polyvinylidene difluoride membrane
1
Exosome Uptake and Protein Expression
2
Molecular Signaling Pathways Analysis
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The collected brain tissues were homogenized with a radioimmunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), and 1% octylphenoxy poly(ethyleneoxy)ethanol (IGEPAL CA-630)) with Protease Inhibitor Cocktail Set I and the Protein Phosphatase Inhibitor Cocktail Set IV (Calbiochem). Equal amounts of lysate samples (20 μg protein) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes (Millipore). The polyvinylidene difluoride membranes were incubated with 2% bovine serum albumin (BSA) in Tris-buffer solution with 0.05% Tween 20 and then with 1 of the following primary antibodies: anti-phospho-CaMKII (Upstate), anti-CaMKII (Millipore), anti-phospho-ser133-CREB (Millipore), anti-CREB (Millipore), or anti-β-actin (Millipore). Horseradish peroxidase-conjugated secondary antibody (Perkin Elmer) was used for all western-blot assays. The membrane was detected with chemiluminescent horseradish peroxidase substrate (Millipore) to visualize the protein bands, which were quantified by NIH Image J software.
3
Western Blot Analysis of Autophagy Markers
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Cardiomyocytes were lysed using cell lysis buffer. Equal amounts of proteins were used for western blotting as described previously [23 (link), 24 (link)]. Briefly, proteins were denatured by boiling, separated by 12% sodium dodecyl sulfate-polyacrylamide gels, and transferred to polyvinylidene difluoride membranes (EMD Millipore, Burlington, MA, USA). Following blocking nonspecific binding with PBS containing 8% nonfat milk and 0.5% Tween-20 for 2 h, the membranes were sequentially incubated with a rabbit antibody against microtubule-associated proteins 1A/1B light chain 3B (LC3B), p62/sequestosome 1, adenosine monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), unc-51 like autophagy activating kinase 1 (ULK1), Beclin-1, phosphorylated-AMPK, phosphorylated-mTOR, phosphorylated-ULK1, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (all antibodies used at 1 : 1000 dilution) and washed and incubated with goat anti-rabbit secondary antibody-horseradish peroxidase conjugate (1 : 3000 dilution). Proteins of interest were visualized using the enhanced chemiluminescence kit, and each band density was quantified by a densitometry. Except for polyvinylidene difluoride membranes and enhanced chemiluminescence kit (EMD Millipore), all reagents and materials were obtained from the Cell Signaling Technology, Danvers, MT, USA.
4
Securin Expression in Stomach Cells
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Cells were collected from the inner layer of the stomach from untreated (n = 2) and RAN + lime- treated mice for 300 days (n = 3). The cells were washed with ice-cold 0.1 M phosphate-buffered saline (PBS; pH 7.4), and total protein was extracted with lysis buffer containing 0.1% SDS, 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 50 mM sodium fluoride, 100 U/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride. After centrifugation, the cell lysate was collected and the protein concentration was determined using the bicinchoninic acid protein assay. Equal amounts of protein (40 µg/well) were subjected to Novex Tris–Glycine 4–20% gradient gels, and electrophoresis was performed in a NuPAGE electrophoresis system (Invitrogen, California, USA). Then the proteins were transferred to a polyvinylidene difluoride membrane (Sigma) and probed with 1:1000 dilution of a mouse monoclonal antibody against securin (DCS-280; ab3305; Abcam, California, USA) and β-actin (AC-15; ab6276; Abcam, USA). Alkaline–phosphatase conjugated anti-mouse IgG (Abcam, USA) was used as the secondary antibody, and immunodetection was performed by treating the blot with the substrate solution of BCIP/NBT (Bangalore Genei, India).
5
Western Blot Analysis of β-Catenin
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Cell extracts were harvested from the SP and non-SP cells using RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) and protein concentration was determined using a Bradford assay (Sigma-Aldrich) (18 (link)). Protein lysates (40 μg) from each sample were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Sigma-Aldrich). Separated proteins were transferred to a polyvinylidene difluoride membrane (Sigma-Aldrich). The membranes were treated with the primary antibodies against β-catenin (1:2,400; cat. no. ab6302; Abcam, Shanghai, China) and GAPDH (0.7 μg/ml; cat. no. ab37168, Abcam). Subsequently, the membranes were incubated in the secondary antibodies horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG with alkaline phosphatase markers; cat. no. ab97048, Abcam). The protein was detected using chemiluminescence reagents (Amersham Biosciences). Blots were scanned using a Bio-Rad GS-710 densitometer (Bio-Rad Laboratories, Inc.).
6
Protein Extraction and Western Blot Analysis
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Cells were lysed with modified RIPA buffer (50 mM of Tris, 150 mM of NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) containing 25 µg/ml of leupeptin, 1 mM of sodium orthovanadate, 2 mM of EDTA and 1 mM of phenylmethylsulfonyl fluoride. The concentration of the protein samples was determined using a BCA kit (Beyotime Institute of Biotechnology, Beijing, China). A total of 20 µg of each protein sample was loaded onto an 8% SDS-PAGE gel, electrophoresed and blotted onto a polyvinylidene difluoride membrane (Sigma-Aldrich; Merck KGaA). The membranes were blotted with the first antibody (anti-p-p38) overnight at 4°C (cat. no. AM063; Beyotime, Zhejiang, China), anti-IDO (cat. no. H00003620-B01P; Abnova, Walnut, CA, USA), anti-Ku80 (cat. no. BS2692; Bioworld Technology, Inc., St. Louis Park, MN, USA) and anti-β-actin antibody (cat. no. MAB12983; Abnova) and with a secondary antibody at room temperature for 1 h. Immunoreactivity was detected using an enhanced chemiluminescence system (Xi'an Jiaotong University) and normalized to β-actin.
7
Quantitative SLO Detection in Bacterial Supernatant
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Equal amounts of bacterial supernatants (7.5 µl), each harvested after reaching precisely OD540 of 1, were loaded on SDS-PAGE gel. Equal loading (total protein) was verified by Coomassie Blue staining.
Liposome-bound SLO or SLO from complete supernatant was detected by Western blot. Recombinant SLO and BHI 10% FBS medium were used as positive and negative control respectively. Immunoblotting was performed with a polyvinylidene difluoride membrane (Sigma-Aldrich, USA). A rabbit polyclonal antibody against SLO (Bio Academia, Japan) was used at 1:2000 dilution. A goat anti-rabbit IgG horseradish peroxidase-linked (BD Bioscience, USA), diluted 1:1000, was used as secondary antibody. The membrane was developed with WesternBright ECL (Advansta, USA), read by Fusion FX (Vilber Lourmat, France), the band intensities were analyzed by FIJI [42 (link)]. The statistical analysis was performed with GraphPad Prism (USA) using a one-way ANOVA followed by post hoc Tukey HSD test.
Liposome-bound SLO or SLO from complete supernatant was detected by Western blot. Recombinant SLO and BHI 10% FBS medium were used as positive and negative control respectively. Immunoblotting was performed with a polyvinylidene difluoride membrane (Sigma-Aldrich, USA). A rabbit polyclonal antibody against SLO (Bio Academia, Japan) was used at 1:2000 dilution. A goat anti-rabbit IgG horseradish peroxidase-linked (BD Bioscience, USA), diluted 1:1000, was used as secondary antibody. The membrane was developed with WesternBright ECL (Advansta, USA), read by Fusion FX (Vilber Lourmat, France), the band intensities were analyzed by FIJI [42 (link)]. The statistical analysis was performed with GraphPad Prism (USA) using a one-way ANOVA followed by post hoc Tukey HSD test.
8
Quantification of VEGF Protein Expression
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The treated cells were lysed with radioimmunoprecipitation assay protein lysis buffer (containing protease inhibitors) and centrifuged at 12,000 × g or 4 min at 4°C. The protein concentration of the supernatant was detected with a bicinchoninic acid assay kit (BioVision). A total of 40 mg of the total protein samples were loaded onto 12% SDS-PAGE gel, separated by electrophoresis and transferred onto a polyvinylidene difluoride membrane (Sigma-Aldrich; Merck KGaA). The membranes were blocked with Tris-buffered saline containing 5% skimmed milk for 1 h, incubated with VEGF antibody (1:500; cat. no. 251622; ZEN-Biotech) overnight at 4°C, triple-washed with Tris-buffered saline containing Tween-20, reacted with secondary antibody (1:500; cat. no. 220173; ZEN-Biotech Pvt. Ltd.) at room temperature for 1 h, triple washed again, and visualized using an enhanced chemiluminescence detection kit (GE Healthcare). The protein bands were then exposed to X-ray film (Fujifilm).
9
Purification and Identification of HPr Protein
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Expression and purification of LsrK via the GST affinity column chromatography and thrombin digestion followed our previously published methods (21 (link)). To identify the copurified protein [that migrated between 10- and 15-kDa size markers during SDS–polyacrylamide gel electrophoresis (SDS-PAGE) (16%) analysis], we performed (i) mass spectrometric analysis and (ii) N-terminal sequencing. The band within SDS-PAGE containing the unknown copurified protein was excised and then in-gel–digested using N-tosyl-l -phenylalanine chloromethyl ketone (TPCK)–treated trypsin. The resulting peptides were analyzed by using an ultrafleXtreme MALDI-TOF mass spectrometer (Bruker Daltonics). Two peptide fragments (M1 to K24 and L50 to K79) were matched to the sequence of the HPr protein. For N-terminal sequencing, the same protein band in SDS-PAGE was transferred into a polyvinylidene difluoride membrane (Sigma-Aldrich), and then the first seven N-terminal amino acids from F2 to I8 of the HPr protein were confirmed using Edman degradation.
10
Analysis of Esophageal and Gastric Cells
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Cells were collected from the inner layer of esophagous and stomach from untreated (n = 5), RAN + lime and PRE + RAN + lime treated mice for 180 and 260 (n = 5 per point) days. In case of 180 days treatment with ECGU + RAN + lime 4 mice were used as a treated group. The cells were washed with ice-cold 0.1 M phosphate-buffered saline (PBS; pH 7.4) and total protein was extracted with a lysis buffer containing 0.1% SDS, 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 50 mM sodium fluoride, 100 U/ml aprotinin and 1 mM phenylmethylsulfonyl fluoride. After centrifugation, the cell lysate was collected and the protein concentration was determined using the bicinchoninic acid protein assay. Equal amount of protein (40 µg/well) were subjected to Novex Tris-Glycine 4–20% gradient gels and electrophoresis was performed in NuPAGE electrophoresis system (Invitrogen, USA). Then the proteins were transferred to a polyvinylidene difluoride membrane (Sigma) and probed with 1:1000 dilution of a mouse monoclonal antibody against p53 (PAb 240; ab-26; Abcam, USA), Securin (DCS-280; ab3305; Abcam, USA) and β-actin (AC-15; ab6276; Abcam, USA). Alkaline–phosphatase conjugated anti-mouse IgG (Abcam, USA) used as secondary antibodies and immunodetection was performed by treating the blot with the substrate solution of BCIP/NBT (Bangalore Genei, India).
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