Total kidney tissue protein was obtained with RIPA buffer as previously described. Protein concentrations were determined with a Bio-Rad protein assay kit (Hercules, CA, USA). For the western blot analysis, aliquots of the lysate containing 30–50 μg protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrotransferred onto a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA). The membranes were subjected to immunoblot analysis and the proteins were visualized by an enhanced chemiluminescence (ECL) method (GE Healthcare, Buckinghamshire, UK). The cell lysates were separated by 12% SDS-PAGE, transferred onto a polyvinylidene fluoride membrane (GE Healthcare, Buckinghamshire, UK), blocked with 5% skimmed milk and hybridized with primary antibodies (diluted 1:1000). The antibodies against NF-κB-p65, IκB-α, TGF-β1, Fas and FasL were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The blots were then incubated with the horseradish peroxidase—conjugated secondary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) for 1 h at room temperature. The blots were washed three times with PBS-T and then developed by enhanced chemiluminescence (Amersham Life Science, Arlington Heights, IL, USA) [12 (link)].
Polyvinylidene fluoride membrane
Manufactured by GE Healthcare
Sourced in United Kingdom, United States, Sweden, Japan
Polyvinylidene fluoride (PVDF) membranes are a type of laboratory equipment used for various applications in biochemistry and analytical chemistry. They are a polymer-based material known for their chemical and thermal resistance, as well as their ability to effectively retain and separate biomolecules. PVDF membranes are commonly used in techniques such as Western blotting, protein and DNA transfer, and sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Protein assay kit, by Bio-Rad (10 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (10 mentions)
Protease inhibitor cocktail, by Roche (9 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (7 mentions)
Bradford assay, by Bio-Rad (6 mentions)
Enhanced chemiluminescence ecl method, by GE Healthcare (5 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (5 mentions)
Enhanced chemiluminescence, by Cytiva (4 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Las 3000, by Fujifilm (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
β actin, by Merck Group (4 mentions)
Nitrocellulose membrane, by GE Healthcare (4 mentions)
Peroxidase conjugated anti rabbit igg, by Merck Group (3 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (3 mentions)
Ecl reagent, by Cytiva (3 mentions)
Enhanced chemiluminescence kit, by GE Healthcare (3 mentions)
Enhanced chemiluminescence, by GE Healthcare (3 mentions)
Phosstop, by Roche (3 mentions)
178 protocols using polyvinylidene fluoride membrane
1
Western Blot Analysis of Kidney Proteins
2
Quantification of His-Tagged hCLDN-4 Proteins
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Samples of His‐tagged hCLDN‐4 with or without sodium dodecyl sulfate and 2‐mercaptethanol (each containing 50 μg protein in 2 μL of phosphate‐buffered saline) were spotted onto polyvinylidene fluoride membrane (GE Healthcare). After blocking of the membranes with Tris‐buffered saline containing 5% skim milk and 0.05% Tween‐20, the membranes were treated with 1 μg/mL of 5D12 or 100‐fold‐diluted mouse anti‐His tag antibody (Life Technologies) as a primary antibody at 4°C for 12 h and then with 1000‐fold‐diluted horseradish‐peroxidase–conjugated anti‐rat IgG (Jackson ImmunoResearch, West Grove, PA) or horseradish‐peroxidase–conjugated anti‐mouse IgG (Jackson ImmunoResearch), respectively, as a secondary antibody at room temperature for 1 h. After the membrane had been washed with Tris‐buffered saline containing 0.05% Tween‐20, the antibody‐reacted dots were detected by using a chemiluminescence imaging system (ECL Western Blotting Detection Reagent and LAS‐4010 ImageQuant, GE Healthcare).
3
Exosomal Protein Verification by Western Blot
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To verify the isolation of exosomes from the serum, western blot analysis for CD9 and CD63, which are enriched in exosomes, was performed. Total proteins were extracted using lysis buffer (Pro-Prep, iNtRON Biotechnology, South Korea) and 20 μg of protein was separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (GE Health care, Piscataway, NJ). After blocking with 5% skimmed milk for 1 hour at room temperature, membranes were incubated overnight at 4°C with primary antibody (anti-β-actin 1:1000 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-CD9 1:1000 (Cell Signaling, Danvers, MA, USA) or anti-CD63 1:1000 (Abcam, Cambridge, UK)) followed by horseradish peroxidase-conjugated anti-mouse 1:1000 or anti-rabbit secondary antibody 1:1000 (Novus Biologicals, Littleton, CO, USA), and incubated for 1 hour at room temperature. After incubation, membranes were washed and proteins revealed by Western Blotting Luminol Reagent (Bio-Rad, Hercules, CA, USA).
4
CFTR Expression in F508del HeLa Cells
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HeLa‐F508del cells were treated 24 h with VX‐809 (10 μM). Total protein fraction was extracted with RIPA buffer and Complete Mini EDTA‐free protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). For Western blots, equal amounts of proteins were resolved by 7% SDS‐PAGE and transferred onto polyvinylidene fluoride membrane (GE Healthcare, LittleChalfont, UK). The membranes were probed with specific antibodies anti‐CFTR 24‐1 (1:200) (R&D Systems, Minneapolis, MN, USA) and anti‐β actin (A5441; 1/10,000) (Sigma‐Aldrich, St‐Louis, USA) and detected with horseradish peroxidase (HRP)‐conjugated goat anti‐mouse IgG (1:50,000) (Sigma–Aldrich, St‐Louis, USA). The signal was detected using the enhanced chemiluminescence ECL Advance kit (GE Healthcare) and visualized using the G:BOX‐iChemi (Syngene, SynopticsLTD, UK).
5
Western Blot Protein Detection Protocol
6
Western Blot Analysis of Apoptosis Regulators
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Total cell lysates were obtained with an extraction buffer as previously described. Protein concentrations were determined using a protein assay kit (Bio-Rad, Hercules, CA, USA). For Western blot analysis, the cell lysates were separated by 12% SDS-PAGE, transferred into a polyvinylidene fluoride membrane (GE Healthcare), blocked with 5% skim milk, and incubated with the primary antibodies (1:1000 dilution). Antibodies against caspase-3, -8, -9, Bax, Bcl-2, Bcl-xL, HIAP-1, HIAP-2, p53, p21, E2F1 and p73 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). After incubation with the horseradish peroxidase-conjugated secondary antibody at room temperature, immunoreactive proteins were detected using a chemiluminescent enhanced chemiluminescence assay kit (GE Healthcare) according to the manufacturer’s instructions. Bands in the blot were visualized using a LAS3000 luminescent image analyzer (Fujifilm Life Science, Tokyo, Japan) [15 (link)].
7
SDS-PAGE and Western Blot Analysis
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Proteins were separated through SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (GE Healthcare) by using a semi-dry transfer unit (Bio-Rad). The membranes were blocked in TBST containing 5% non-fat milk and 0.1% Tween-20, incubated with the indicated primary antibodies for 2 h at room temperature, and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Immunoreactivity was visualized with an ECL chemiluminescence system (Santa Cruz).
8
Antibody Production and Western Blot Analysis
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The purified proteins psaDb (CISIN_1g028581mg) and GBSS1 (CISIN_1g007224mg) were used as antigens for antibody production. Antibodies were produced in rabbit by Huaan Biotechnology Company (Hangzhou, China). For Western blot analysis, the total protein of Citrus petioles was extracted and the implementation method refers to Section 2.3. Total proteins (20 μg) from each sample were resolved on 12% SDS-PAGE, and the protein was transferred onto a polyvinylidene fluoride membrane (GE Healthcare, Chicago, Illinois, United States). The blots were analyzed using respective primary antibodies at a dilution of 1:500 in PBS. Protein bands were visualized using BeyoECL Star kit (Beyotime, Shanghai, China).
9
Protein Turnover Dynamics Analysis
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The turnover of ΔssCPY∗-GFP and Hmg2-myc was assessed in cells by inhibiting protein synthesis with cycloheximide. Cells were collected at the indicated time points and ΔssCPY∗-GFP and Hmg2-myc levels analyzed by immunoblotting and quantified by densitometry. Values shown are percentages relative to zero time points from three independent biological repeats. Protein extracts were electrophoresed under reducing conditions on NuPAGE minigels (Thermo Fisher Scientific) and electroblotted onto polyvinylidene fluoride membrane (GE Healthcare). Primary antibodies used were rabbit α-Sup35 (Ness et al., 2002 (link)), ubiquitin (sc-8017; Santa Cruz Biotechnology, Inc.), Ydj1 (ab74442; Abcam), Sis1 (COP-080051; Operon Biotechnologies), Ssa1 (ADI-SPA-822; Enzo Life Sciences), GFP (A6465, Invitrogen), Pgk1 (459250; Thermo Fisher Scientific), Hsp104 (ab2924; Abcam), rabbit α-Sse1 (Chiabudini et al., 2012 (link)), and Myc 4A6 (05–724; EMD Millipore).
10
Western Blot Analysis of SLP-2 Protein
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Cells and ground frozen tissues were harvested and lysed in sampling buffer [62.5 mmol/l Tris-HCl (pH 6.8), 2% SDS, 10% glycerol and 5% 2-h-mercaptoethanol]. Protein concentration was determined using a Bradford assay (Bio-Rad Laboratories, Inc.). A total of 20 µg of proteins were separated by 10% SDS-PAGE, prior to being transferred onto a polyvinylidene fluoride membrane (GE Healthcare Life Sciences, Chalfont, UK). Subsequent to being blocked in 5% non-fat dry milk, the membrane was incubated for 12 h at 4°C with an anti-SLP-2 rabbit polyclonal antibody (dilution, 1:3,000; cat. no. AP20280c; Abgent, Inc., San Diego, CA, USA), then the membranes were washed with PBST and exposed to a horseradish peroxidase-conjugated anti-rabbit secondary antibody (dilution, 1:2,000; cat. no. NA934; GE Healthcare Life Sciences) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence kit (GE Healthcare Life Sciences). An anti-a-tubulin polyclonal antibody (dilution, 1:1,000; cat. no. SAB4500087; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) was used as the loading control.
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