Equal amounts of protein were subjected to SDS-PAGE and then transferred to a PDVF membrane (Bio-Rad, Hercules, CA). Subsequently, membranes were incubated with primary antibodies and HRP-conjugated secondary antibody (Cell Signaling, Beverly, MA). Proteins were revealed by using ProSignal® Dura ECL Reagent (Genesee Scientific, San Diego, CA) and visualized in a Chemidoc™ Touch Imaging System (Bio-Rad).
Pdvf membrane
Manufactured by Bio-Rad
Sourced in United States, United Kingdom
PVDF membranes are a type of laboratory equipment used for protein transfer and detection. They provide a stable and durable surface for the immobilization of proteins, allowing for their subsequent analysis and identification.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc, by Bio-Rad (3 mentions)
Bicinchoninic acid (bca), by Thermo Fisher Scientific (3 mentions)
Sds page, by Bio-Rad (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Mini protean tgx precast gel, by Bio-Rad (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (3 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Hrp conjugated secondary antibody, by Cell Signaling Technology (2 mentions)
Prosignal dura ecl reagent, by Genesee Scientific (2 mentions)
Ecl reagent, by Bio-Rad (2 mentions)
Molecular imager chemidoc xrs, by Bio-Rad (2 mentions)
Tbs t, by Cell Signaling Technology (2 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (2 mentions)
Amersham ecl, by Cytiva (2 mentions)
Pdvf membrane, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Merck Group (2 mentions)
Restore plus western blot stripping buffer, by Thermo Fisher Scientific (2 mentions)
49 protocols using pdvf membrane
1
Western Blot Protein Detection
2
Western Blot Protein Analysis Protocol
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Protein lysates (20-25μg) were denatured using 1X Laemmli Buffer and run on 7.5, 10, or 16% SDS PAGE gels at 100V for about 2 hours. Proteins were then transferred onto a PDVF membrane (BioRad or Thermo Fisher Scientific) at 100V for 1 hour. Ponceau-S staining was done to verify transfer. Non-specific binding was blocked by incubating the membrane for at least one hour at room temperature or overnight at 4°C in 5% milk in TBS for non-phosphorylated proteins, or 5% Bovine serum albumin (BSA Fraction V, Fisher Scientific) in TBS for phosphorylated proteins. The membrane was then blotted with primary antibody diluted in 5% BSA or Milk in TBS supplemented with 0.1% Tween 20 (TBST). After an overnight incubation at 4°C, the membrane was washed with TBST and incubated in the secondary antibody for at least 2 hours at room temperature in 5% BSA or Milk in TBST. After washing out the secondary antibody in TBST, the membrane was exposed to BioRad's Clarity Western ECL substrate according to manufacturer's instructions. Signal was detected using UltraCruz Autoradiography Film (Santa Cruz Biotech). Antibodies for GAPDH and p-IκBα were obtained from Cell Signaling (Catalog # 14C10 and 14D4, respectively). BCL2α, Pro-caspase 3, PARP-1, and cytochrome c antibodies were obtained from Santa Cruz Biotech Catalog # SC-7382, SC-7148, SC-8007, and SC-271627, respectively.
3
Western Blot Protein Analysis Protocol
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Cells were lysed with Pierce RIPA buffer (Thermo Fisher) supplemented with Halt™ Protease and Phosphatase Inhibitor (Thermo Fisher) on ice for 30 min. Samples were quantified by Pierce™ BCA assay (Thermo Fisher). Samples of at least 20 µg of protein were run on tris‐glycine polyacrylamide gel electrophoresis (PAGE) gel. Proteins from the gel were transferred to PDVF membrane (Bio‐Rad) overnight at 150 mA at 4°C. Membranes were blocked for at least 30 min in 5% milk/ tris‐buffered saline with 0.1% tween (TBST) and incubated in primary antibody (5% milk/TBST) overnight at 4°C. The following day, membrane was incubated with an anti‐rabbit HRP‐conjugated (GE) secondary antibody for 1 h at room temperature (RT). Chemiluminescent reagents (Millipore) were applied to the membrane, and Western blot image was developed by film developer (Konica Minolta Film Processor) using film (Konica Minolta).
4
SDS-PAGE Protein Detection using Streptavidin-Alexa488
5
Western Blot Protein Detection
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Equal amounts of protein were subjected to SDS-PAGE and then transferred to a PDVF membrane (Bio-Rad, Hercules, CA). Subsequently, membranes were incubated with primary antibodies and HRP-conjugated secondary antibody (Cell Signaling, Beverly, MA). Proteins were revealed by using ProSignal® Dura ECL Reagent (Genesee Scientific, San Diego, CA) and visualized in a Chemidoc™ Touch Imaging System (Bio-Rad).
6
Western Blot Analysis of Protein Samples
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Post lysis, proteins were separated by SDS-PAGE (200 V, 45 min) before being transferred to a PDVF membrane (Bio-Rad) with a Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, 20 V, 1 h). Western blots were blocked in OneBlock™ Western-CL blocking buffer (Genesee) for 1 h rt. Blots were then incubated with primary antibody (1:1,000 dilution) in fresh blocking buffer at 4 °C overnight. Following overnight incubation, blots were washed in TBST (Cell Signaling, 3× 10 min) and incubated with (HRP)-conjugated secondary antibody (1:10,000 dilution) in fresh blocking buffer for 1 h at rt. Once complete, blots were again washed in TBST (3× 10 min). Blots were developed using ECL reagents (Bio-Rad) and the ChemiDoc XRS+ molecular imager (Bio-Rad, Bio-Rad Image Lab 4.1).
7
Protein Analysis of Mouse Skin Wound Healing
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Normal mouse skin tissue and mouse wound tissue 7 and 23 days post wounding were lysed in radioimmunoprecipitation (RIPA) buffer supplemented with a phosphatase inhibitor (PhosSTOP, Roche). The lysates were incubated for 20 min on ice and centrifuged at 20,000 × g for 15 min at 4°C. The supernatants were denatured at 95°C for 5 min in SDS sample buffer, consisting of 62.5 mmol/L Tris (pH 6.8), 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, and 0.001% bromophenol blue. Samples were separated by SDS–PAGE, and proteins were transferred onto a PDVF membrane (Bio-Rad, Munich, Germany). Membranes were incubated for 30 min at room temperature in blocking buffer consisting of 5% nonfat dry milk in phosphate-buffered saline (PBS) with 0.05% Tween-20, followed by an appropriate dilution of anti-POSTN Ab (Abcam) or anti-α-tubulin (Sigma) primary antibody overnight at 4°C. Immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Piscataway, NJ), a chemiluminescence detection system (Perkin-Elmer, Waltham, MA), and a LAS-3000 instrument (Fujifilm, Tokyo, Japan)
8
Western Blot Analysis of Aortic α1-Adrenergic Receptor and eNOS
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Dissected aortas were homogenized with buffer containing 50 mmol/L Tris‐HCl (pH7.4), 150 mmol/L NaCl, 1% Nonidet P‐40, 1 mmol/L EDTA, 1% protease inhibitor cocktail (P8340) (Sigma‐Aldrich, Darmstadt, Germany), and 1% phosphatase inhibitor cocktail (167‐24381) (Wako Pure Chemical Industries, Osaka, Japan). The homogenate containing 100 μg (for α1 adrenergic receptor) or 50 μg protein (for eNOS) was separated by 6% SDS‐polyacrylamide gel electrophoresis, and transblotted to PDVF membrane (Bio‐Rad, Hercules). α1 Adrenergic receptor was detected by ab3462 (Abcam, Cambridge, UK) and eNOS was detected by 610296 (BD Science, Franklin Lakes) as a primary antibody, respectively. The membranes were striped and reincubated with anti‐β‐actin antibody (AC‐15) (Sigma‐Aldrich, St Louis) for normalization. Signals were detected by horse radish peroxidase‐conjugated secondary antibodies and ECL select (GE Healthcare Life Science, Buckinghamshire, UK). The density of the signals was then quantified by LAS 4000mini using ImageQuant TL software (GE Healthcare Life Science Buckinghamshire, UK).
9
Western Blot Quantification Protocol
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Cells were lysed on ice with NP-40 lysis buffer containing protease inhibitor cocktail (Life Technologies) and stored in a −20 freezer. Lysates were adjusted for protein concentration with the Bradford assay according to the manufacturer’s protocol (BioRad, Copenhagen, Denmark), and 50 µg of protein separated by 4–20% SDS-PAGE, and blotted on PDVF membrane (BioRad, Copenhagen, Denmark). The membranes were stained with anti-IkB (Santa Cruz, Heidelberg, Germany) and alpha-tubulin (Sigma, Copenhagen, Denmark) antibodies and developed with the chemiluminescence detection system Super Signal (Life Technologies, Naerum, Denmark) as previously described [45 (link)]. Light emission was captured using an Alphaimager system (Alpha Innotech, MultiImage III, Broager, Denmark). Band density was quantified using ImageJ software.
10
Exosome Isolation and Characterization
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Transfection was performed with pLV-EF1α-GFP-miR9T or pLV-EF1α-mEmerald-CD9-miR9T plasmid (2.5 μg/well) using Lipofectamine 3000 (Thermo Fisher Science, #L3000008). Whole cells and conditioned medium were collected 3 d after transfection. The conditioned medium was centrifuged at 2000 g for 30 min, at 10,000 g for 30 min, at 144,000 g for 70 min and last pellets were collected as the EV fraction. Whole cells or EV fractions were lysed with RIPA buffer containing Halt Protease and Phosphatase inhibitor Cocktail (Thermo Fisher Science, #78442), and incubated at 95 °C for 5 min with Laemmli sampling buffer (Bio-Rad, #S3401-10VL) supplemented with 2% v/v 2-mercaptoethanol. The protein samples (10 μg/lane) were subjected to 10% SDS-PAGE, transferred to PDVF membrane (Bio-Rad), and blocked with 3% skim milk in TBS 0.5% Tween20 (TBS-T). The membranes were incubated overnight at 4 °C with anti-GFP mouse monoclonal antibody (Santa Cruz Biotech, B-2; 1:500), and washed with TBS-T. Then the membranes were incubated with HRP-conjugated anti-mouse IgG antibody (Cell Signaling Technology, #7076; 1:10,000) for 1 h at room temperature. The proteins were detected with Immobilon chemiluminescent HRP substrate (Millipore Sigma, #WBKLS0100) and visualized with Chemiluminescent Western Blot Imaging System (Azure, #C300).
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