Cells were lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA, 0.1% sodium dodecyl sulfate, 1 mM sodium vanadate, 1 mM NaF, 1 mM phenylmethanesulfonyl fluoride, 0.1 mg/ml pepstatin, 0.1 mg/ml leupeptin, and 0.1 mg/ml aprotinin). The protein concentration was determined by using a bicinchoninic acid protein assay. Protein lysates (40 μg) were then resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene difluoride membranes (PVDF; Bio-Rad, Hercules, CA, USA), and blotted with different primary antibodies [anti-IL-18; Abcam, Cambridge, UK; anti-GSK-3β, anti-phosphorylated GSK-3β (p-GSK-3β), anti-caspase-3, anti-cleaved caspase-3, anti-caspase-7, anti-cleaved caspase-7; all from Cell Signaling Technology, Boston, MA, USA] overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies and visualized with an ECL reagent (GE Healthcare, London, UK).
Polyvinylidene difluoride membranes pvdf
Manufactured by Bio-Rad
Sourced in United States
Polyvinylidene difluoride membranes (PVDF) are a type of membrane material used in various laboratory applications. PVDF membranes are known for their chemical and thermal resistance, as well as their ability to effectively bind and transfer proteins, nucleic acids, and other biomolecules during analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Anti proliferating cell nuclear antigen, by Merck Group (2 mentions)
Anti β actin immunoglobulin, by Merck Group (2 mentions)
Goat anti mouse igg, by Bio-Rad (2 mentions)
Anti cleaved caspase 7, by Cell Signaling Technology (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Anti gsk3β, by Cell Signaling Technology (1 mentions)
Anti phosphorylated gsk 3β p gsk 3β, by Cell Signaling Technology (1 mentions)
Anti il 18, by Abcam (1 mentions)
Anti caspase 7, by Cell Signaling Technology (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
Ab138515, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Sox12, by Abcam (1 mentions)
Pierce bicinchoninic acid bca protein detection kit, by Bio-Rad (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Ecl solution, by Bio-Rad (1 mentions)
Image pro plus 6.0 ipp software, by Media Cybernetics (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
5 protocols using polyvinylidene difluoride membranes pvdf
1
Apoptosis Signaling Pathway Analysis
2
Vascular Reactivity Assay Protocol
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HCB (>99% purity), phenylephrine (Phe), acetylcholine (Ach) and nitroprusside (SNP) were purchased from Sigma-Aldrich Co (St Louis, MO, USA). Reverse transcriptase, deoxynucleotide triphosphates (dNTPs), random primers, Taq enzyme polymerase and molecular weight markers were purchased from Biodynamics SRL (Buenos Aires, Argentina). Primers for TGF-β1 and ribosomal protein L19 (L19) were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Anti-proliferating cell nuclear antigen (PCNA) (code: A3271) and anti-β-actin immunoglobulin (code: A1978) were from Sigma-Aldrich Co. Anti-AT1 (code: ab124734) and anti- endothelial nitric oxide synthase (eNOS) (code: ab252439) were purchased from Abcam Inc. (Cambridge, UK). Anti-rabbit IgG (code: 156831), goat anti-mouse IgG (code: 31430), and polyvinylidene difluoride membranes (PVDF) were purchased from Bio-Rad Laboratories Inc. (Hercules, CA, USA). CP-BU plates were purchased from Agfa Gevaert S.A. (Buenos Aires, Argentina). Losartan was generously provided by GADOR S.A. (Buenos Aires, Argentina). All other reagents were of molecular biology grade and at least 99% purity.
3
Western Blot Analysis of EMT Markers
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Radioimmunoprecipitation assay (RIPA) lysis buffers (CST, Danvers, MA, USA) containing proteinase and phosphatase inhibitors were used to lyse the transfected MHCC97-H, HCCLM3, and Hep 3B cells. After being centrifuged for 30 min, a Pierce Bicinchoninic acid (BCA) Protein detection kit (Bio-Rad Laboratories, Hercules, CA, USA) was used for assessing the concentration of the proteins. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) used to separate proteins in each sample, and the proteins were then transferred to the polyvinylidene difluoride membranes (PVDF; Bio-Rad Laboratories, Hercules, CA, USA). The membranes were disposed with 5% non-fat milk and then cultured with primary antibodies E-cadherin (#3195, 1:1,000; CST), N-cadherin (#13116, 1:1,000; CST), Vimentin (#5741, 1:1,000; CST), GAPDH (ab9485, 1/10,000; Abcam), β-catenin (#8480, 1:1,000; CST), c-Myc (#18583, 1:1,000; CST), Cyclin D1 (#55506, 1:1,000; CST), MMP-7 (#71031, 1:1,000; CST), β-actin (#4970, 1:1,000; CST), E2F2 (ab138515, 1/1,000; Abcam), and SOX12 (cat. no. 23939-1-AP, 1:500; Proteintech) at 4°C overnight. Later, membranes were cultured with secondary antibodies (ab205718, 1/10,000; Abcam) at the room temperature for 1 h. The bands were visualized via enhanced chemiluminescence (ECL; Millipore, USA). GAPDH or β-actin was employed as the internal control. Experiments were conducted three times.
4
Western Blot Analysis of CXCR7 Expression
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Transfected cells were incubated in radioimmunoprecipitation assay (RIPA) buffer (Beyotime Institute of Biotechnology) for 15 min. The protein concentration of the samples was calculated using a bicinchoninic acid (BCA) protein concentration assay kit. Each sample (20 mg/lane) was mixed with loading buffer (Beyotime Institute of Biotechnology) and boiled for 5 min at 95°C in the heating module. Then, 20 mg per lane of proteins from each sample were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), later transferred to polyvinylidene difluoride membranes (PVDF; Bio-Rad Laboratories, Inc.). The membranes were blocked at room temperature for 2 h with 3% bovine serum albumin (BSA, Hyclone; GE Healthcare Life Sciences) and washed them three times with TBST. The dilution ratio for the anti-CXCR7 antibody (cat. no. ab72100) was 1:1,000 and for the anti-GAPDH antibody (cat. no. ab181602) was 1:2,000 (both Abcam). The primary antibody was incubated overnight at 4°C. After several washings, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (anti-rabbit) (1:2,000 dilution) for 2 h at 37°C. The protein bands were developed by adding ECL solution (Bio-Rad Laboratories, Inc.). The levels of protein expression were evaluated by Image Pro Plus 6.0 (IPP) software (Media Cybernetics, Inc.).
5
Analysis of TGF-β1 and related proteins
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HCB (>99% purity) was purchased from Sigma-Aldrich Co. The reagents used for cDNA synthesis, reverse transcriptase, molecular weight markers, random primers, desoxi-nucleotide triphosphate (dNTPs), and Taq enzyme polymerase, were purchased from Biodynamics SRL (Buenos Aires, Argentina). Specific primers for TGF-β1 and glyceraldehyde 3-phosphate dehydrogenase (GPDH) were purchased from Invitrogen Life Technology (Carlsbad, CA).
Polyvinylidene difluoride membranes (PVDF), goat anti-mouse IgG, and anti-rabbit IgG were purchased from Bio-Rad Laboratories Inc. CP-BU plates were purchased from Agfa, (Gevaert, Argentina S.A). Anti-β-actin immunoglobulin and anti-proliferating cell nuclear antigen (PCNA) from Sigma-Aldrich Co., monoclonal anti-TGF-β1, anti-AT1 and anti-eNOS from Abcam Inc.. All reagents present purification degree of 96% and molecular biology grade.
Polyvinylidene difluoride membranes (PVDF), goat anti-mouse IgG, and anti-rabbit IgG were purchased from Bio-Rad Laboratories Inc. CP-BU plates were purchased from Agfa, (Gevaert, Argentina S.A). Anti-β-actin immunoglobulin and anti-proliferating cell nuclear antigen (PCNA) from Sigma-Aldrich Co., monoclonal anti-TGF-β1, anti-AT1 and anti-eNOS from Abcam Inc.. All reagents present purification degree of 96% and molecular biology grade.
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