Western blot samples were harvested and lysed either directly in reducing SDS sample buffer (Boston BioProducts BP-111R) or RIPA buffer (Tris-HCl 50 mM pH 7.4, NaCl 150 mM, NP-40 1%, sodium deoxycholate 0.5%, SDS 0.1%) supplemented with protease and phosphatase inhibitors (Leupeptin, Aprotinin, 1 mM phenylmethyl sulphonyl fluoride, Pepstatin, 1 mM sodium fluoride, 1 mM sodium orthovanadate). Lysates were clarified by centrifugation at 13,000g for 10 min and reducing SDS sample buffer was added. The cleared lysates were then boiled in 1 × sample buffer for 5 min and resolved by 4–20% SDS–PAGE gradient gels (Invitrogen), transferred to polyvinylidene difluoride membranes (Whatman), blocked with 5% BSA in TBS (25 mM Tris, 140 mM NaCl, 2.7 mM KCl, pH 7.4), 0.1% Tween 20 for 1 h at room temperature and probed by antibodies overnight. Membranes were subsequently probed with secondary antibodies linked to HRP (GE Healthcare). Western blot membranes were developed using Luminata Western HRP substrate for detection of HRP (Millipore). Western blot results were prepared using Kodak film developer and Epson 3000 scanner. Scanned films of uncropped blots are shown in Supplementary Figs 6–8 .
Polyvinylidene difluoride membrane
Manufactured by Cytiva
Sourced in United Kingdom, United States, Germany, Sweden
Polyvinylidene difluoride (PVDF) membranes are a type of laboratory filter membrane used for various analytical and preparative techniques. They are made of a synthetic polymer material that is chemically and physically resistant, making them suitable for a wide range of applications. PVDF membranes are commonly used for protein and nucleic acid transfer and detection, as well as for sample preparation and filtration processes in biological and chemical research.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (15 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (13 mentions)
Bca kit, by Thermo Fisher Scientific (13 mentions)
β actin, by Santa Cruz Biotechnology (12 mentions)
Enhanced chemiluminescence system, by Roche (10 mentions)
Enhanced chemiluminescence kit, by Cytiva (9 mentions)
Protease inhibitor cocktail, by Merck Group (8 mentions)
Ecl plus kit, by Cytiva (8 mentions)
β actin, by Cell Signaling Technology (7 mentions)
Goat anti rabbit igg, by Santa Cruz Biotechnology (5 mentions)
Gapdh, by Santa Cruz Biotechnology (5 mentions)
Bradford protein assay kit, by Bio-Rad (5 mentions)
Bradford assay, by Bio-Rad (5 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Protein assay kit, by Bio-Rad (4 mentions)
Protein assay, by Bio-Rad (4 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (4 mentions)
Imagequant las 4000 system, by GE Healthcare (4 mentions)
Enhanced chemiluminescence system, by Cytiva (4 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (4 mentions)
249 protocols using polyvinylidene difluoride membrane
1
Western Blot Sample Preparation
2
Protein Expression Analysis in Cells
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Cells and tissues (n = 6) were added to ice-cold RIPA-buffer (Sigma-Aldrich Chemie, Munich, Germany) containing 1 µg/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, 5 mmol/L NaF, 1 mmol/L Na3VO4 and 1 mmol/L dithiothreitol and disrupted in a TissueLyzer (Qiagen, Hilden, Germany). Proteins (40 µg) were separated on 10% (w/v) sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Whatman, Dassel, Germany). Membranes were blocked with PBS containing 0.5% (v/v) Tween 20, 5% (w/v) fat-free milk and 2% (w/v) bovine serum albumin. Protein bands were detected using the primary antibodies anti-SSTR2 [UMB1, ab134152] (Abcam, Cambridge, UK), anti-chromogranin A [C-20, sc1488] (Santa Cruz Biotechnology, Heidelberg, Germany), anti-ß-actin [A5316] (Sigma-Aldrich). The peroxidase-conjugated secondary antibodies anti-mouse IgG [A9044] and anti-rabbit IgG [A0545] were purchased from Sigma-Aldrich. Specific binding was detected using SuperSignal™ West Pico/Dura Substrate (Life Technologies, Carlsbad, CA, USA). Densitometric analysis was performed using the TL100 image analysis software (TotalLab, Newcastle upon Tyne, UK) and normalized to ß-actin.
3
Quantification of miRNA-regulated Proteins
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Proteins were extracted from miR-204-, miR-211-, or miR-negative control-transfected MCF-7 cells using Pierce IP Lysis Buffer (Thermo Fisher Scientific, Waltham, MA, USA) with a protease inhibitor cocktail (Thermo Fisher Scientific). Diluted protein samples were separated on 4–12% gradient SDS-PAGE gels (Koma Biotech, Korea) and transferred to polyvinylidene difluoride membranes (Whatman, UK). Then, the membranes were blocked in 5% non-fat powdered milk (Bio Basic, Canada) for 1 h and incubated for 1 h with anti-MX1 (1:500, Santa Cruz, Dallas, Texas, USA) and anti-TXNIP (1:500, Novus, St. Charles, MO, USA) antibodies. The membranes were then rinsed in 0.01% Tween-20 TBS five times for 5 min each and incubated with horseradish peroxidase conjugated secondary donkey anti-rabbit antibody (1:5000, Amersham, UK) for 1 h. The bands were detected by an EzWay ECL Western Blot Substrate Kit (Koma Biotech) and Image Lab software (Bio-Rad, Hercules, CA, USA). As an endogenous reference, rabbit polyclonal anti-β-actin antibody (1:500) (Novus) was used.
4
Western Blot Analysis of Liver Proteins
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Liver tissue was homogenized (1:9, w/v) with a tissue lysis/extraction reagent (Sigma-Aldrich) containing a protease inhibitor cocktail (Sigma-Aldrich). Equal amounts of protein (40 µg/well) from each sample were separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Whatman, Maidenstone, UK). The membrane was blocked with blocking buffer (5% skim milk) for 1 h at room temperature followed by an overnight incubation at 4℃ with appropriate primary antibodies. The following primary antibodies and dilutions were used: anti-Lamin B1 (1:1000 dilution; Santa Cruz Biotechnology, CA, USA), p38 MAPK, phospho-p38 MAPK (p-p38 MAPK), p44/42 MAPK (Erk1/2), p-Erk1/2, c-Jun amino-terminal kinase (JNK), p-JNK, iNOS, Cox-2, NF-κB p65, TNF-α, and β-actin (1:1000 dilution; Cell Signaling Technology, Beverly, MA, USA). The membrane was washed three times with Tris-buffered saline containing Tween 20 (TBST) and then incubated with horseradish peroxidaseconjugated secondary antibodies (1:2000 dilution; Sigma-Aldrich) for 1 h at room temperature. The blots were washed thrice with TBST and then developed using an enhanced chemiluminescence kit (Thermo Scientific, Waltham, MA, USA). To determine protein expression level, each band density was quantified using TINA 20 Image software (Raytest Isotopenmessgeraete GmbH, Straubenhardt, Germany).
5
Western Blot Analysis of Protein Expression
6
Protein Expression Analysis by Western Blot
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Total protein was isolated from tissues or transfected cells. Protein concentration was determined using BCA protein quantitation kit. Protein (50μg of each sample) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred electrophoretically onto polyvinylidene difluoride membranes (Whatman, Maidstone, UK). Then, the membranes were blocked in 5% skim milk for 1 h, washed three or four times with Tris-buffered saline containing 20% Tween 20 (TBST) at room temperature, and incubated with the primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA): rabbit anti-human RGMB (1:500) or Ki67 (1:500) at 4°C overnight. Following extensive washing with TBST, the membranes were then incubated with secondary antibody (goat anti-rabbit IgG, 1:1000, Santa Cruz Biotechnology) at room temperature for 1 h, washed with TBST four times (15 min each)., the immunoreactivity was visualized by enhanced chemiluminescence. GAPDH (Santa Cruz Biotechnology) served as an endogenous control. The experiment was independently repeated in triplicate.
7
Western Blot Analysis of p53 and GAPDH
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Cells were harvested by scraping from the culture dish and washed twice with ice-cold phosphate buffered saline (PBS). Total protein was extracted from the cell pellet with RIPA lysis buffer (Upstate, Charlottesville, VA). Samples were incubated on ice for 30 min and then centrifuged at 12,000 g for 20 min at 4 °C. Supernatants were collected and mixed with sodium dodecyl sulfate sample buffer, heated at 95 °C for 10 min and separated by polyacrylamide gel electrophoresis and electrical blotted to polyvinylidenedifluoride membranes (Whatman, Inc., Clifton, NJ). Membrane was incubated for 1 h at room temperature in a blocking solution composed of 5% skimmed milk powder dissolved in TBST (0.05% Tween-20, 10 mM Tris, pH 8.0, and 140 mM NaCl). After washing the membrane three times with TBST, proteins were revealed by mouse and rabbit antibodies against p53, or GAPDH by overnight incubation at 4 °C. After three washes with TBST, membranes were incubated with anti-mouse or anti-rabbit secondary antibodies (Cell Signaling Technology, Inc.). The blots were then subjected to chemiluminescent detection according to the manufacturer’s instructions.
8
MERS-CoV-E Protein Expression Verification
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Western blot analysis was performed for verification of MERS-CoV-E protein expression. Transfer of the SDS-PAGE gels was to Polyvinylidene difluoride membranes (Whatman) which had been pre-soaked in 100% methanol (Fisher) for about five minutes.
The gels and membranes were then transferred into 1× transfer buffer (25 mM Tris, 192 mM Glycine pH 8.3 with 20% methanol) using a semi-dry western blotting device, and electroblotted for 80 minutes at 35 V/150 mA. The membranes were then blocked for 1 hour in a solution containing 5% skimmed milk powder, 0.2% tween-20, and 1× TBS. Membranes were incubated for 60-minute with the primary antibody, such as Rabbit polyclonal 6× His tag Ab at 1: 10,000 in 1× TBST buffer and washed three times with TBST buffer for 5 minutes each on a platform rocker. A secondary antibody horseradish-peroxidase (HRP) conjugated goat anti-rabbit antibody (Dako) in a dilution of 1:10,000 in 1× TBST was added to the membranes and incubated for 60 minutes pursued by three times wash with TBST buffer on a platform rocker. According to manufacturer protocol a chemiluminescent reagent (Cyanagen) was used for visualization of protein expression and the membrane imaged using a Syngene G: BOX.
The gels and membranes were then transferred into 1× transfer buffer (25 mM Tris, 192 mM Glycine pH 8.3 with 20% methanol) using a semi-dry western blotting device, and electroblotted for 80 minutes at 35 V/150 mA. The membranes were then blocked for 1 hour in a solution containing 5% skimmed milk powder, 0.2% tween-20, and 1× TBS. Membranes were incubated for 60-minute with the primary antibody, such as Rabbit polyclonal 6× His tag Ab at 1: 10,000 in 1× TBST buffer and washed three times with TBST buffer for 5 minutes each on a platform rocker. A secondary antibody horseradish-peroxidase (HRP) conjugated goat anti-rabbit antibody (Dako) in a dilution of 1:10,000 in 1× TBST was added to the membranes and incubated for 60 minutes pursued by three times wash with TBST buffer on a platform rocker. According to manufacturer protocol a chemiluminescent reagent (Cyanagen) was used for visualization of protein expression and the membrane imaged using a Syngene G: BOX.
9
Immunoblotting for MMP2 and XIAP
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In different cell groups, total proteins were extracted and subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis for separation and then transferred onto polyvinylidene difluoride membranes (Whatman, UK). Blocking was performed in 5% skim milk for 1 h. Then, membranes were washed four times with wash buffer at room temperature and incubated overnight at 4°C with primary antibodies (Santa Cruz, USA). In this study, rabbit anti-human matrix metalloproteinase 2 (MMP2) (1:500) and rabbit anti-human X-linked inhibitor of apoptosis (XIAP) (1:500) were used. The secondary antibody (goat anti-rabbit IgG, 1:1000, Santa Cruz, USA) was incubated for 1 h. After washing, signals from different proteins were determined using a chemiluminescence detection kit (Amersham Pharmacia Biotech, UK). GAPDH (Santa Cruz, USA) served as an endogenous control.
10
Calcium Signaling Pathways in Cellular Stress
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Ang II, Calhex 231 , R568, GdCl 3 , compound C, and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for calcium/calmodulin-dependent protein kinase II (CaMKII), p-CaMKII, p62, Beclin1, microtubule-associated protein light chain 3 (LC3), caspase-3, AMP-activated protein kinase (AMPK), p-AMPK, Ca 2+ /calmodulin-dependent-protein kinase-kinase-β (CaMKK β ), p-CaMKK β , mammalian target of rapamycin (mTOR), p-mTOR, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-CaSR was from Alpha Diagnostic International Inc. (San Antonio, TX, USA). The secondary antibody (alkaline phosphatase-conjugated anti-rabbit IgG) was from Promega Corporation (Madison, WI, USA). Polyvinylidene difluoride membranes were from Whatman (Buckinghamshire, UK).
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