After electrophoresis, the gels were blotted to an Immobilon-P PVDF Membrane (Merck Millipore, Burlington, MA, US) using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad Laboratories) at 200 mA for 80 minutes. After blocking with 1% bovine serum albumin (BSA) (VWR, Radnor, PA, US), the membranes were probed with 1 μg/mL mAb 9G3 against the glycosylated epitope ‘KEPAPTTT’ in the lubricin mucin domain (Merck KGaA, Darmstadt, Germany12 ) or polyclonal anti-CG antibody (Abcam, UK) 1/1000 diluted in assay buffer (1% BSA in PBS-Tween) for detection of endogenous CG in SF, followed by Horseradish Peroxidase (HRP) conjugated goat anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody (Invitrogen, USA) 1/4000 diluted in assay buffer. For the lectin staining assay, blots were first probed by 1 μg/ml biotinylated Peanut Agglutinin (PNA, Vector laboratories, CA,USA) followed by 0.2 μg/ml HRP-streptavidin (Vector laboratories). After incubations, membranes were stained by WesternBright ECL Spray (Advansta, USA) and visualized in a luminescent image analyser (LAS-4000 mini, Fujifilm, Japan). Band intensities were calculated by ImageJ 1.50i53 (link).
Immobilon p pvdf membrane
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Ireland, Australia, Italy, Morocco
Immobilon-P PVDF membrane is a polyvinylidene difluoride (PVDF) membrane designed for protein transfer and immobilization in Western blotting and other protein analysis applications. The membrane provides high protein-binding capacity and is compatible with a variety of detection methods.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (48 mentions)
Protease inhibitor cocktail, by Roche (24 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (22 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (18 mentions)
Image lab software, by Bio-Rad (17 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (16 mentions)
Ripa buffer, by Thermo Fisher Scientific (16 mentions)
β actin, by Merck Group (15 mentions)
Chemidoc mp imaging system, by Bio-Rad (14 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (14 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (13 mentions)
Protease inhibitor cocktail, by Merck Group (13 mentions)
Clarity western ecl substrate, by Bio-Rad (12 mentions)
Complete protease inhibitor cocktail, by Roche (11 mentions)
Ripa buffer, by Merck Group (11 mentions)
Laemmli sample buffer, by Bio-Rad (11 mentions)
Chemidoc touch imaging system, by Bio-Rad (11 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (10 mentions)
Dc protein assay, by Bio-Rad (10 mentions)
Dc protein assay kit, by Bio-Rad (10 mentions)
666 protocols using immobilon p pvdf membrane
1
Western Blot Analysis of Lubricin and CG
2
Protein Expression Analysis by Immunoblot
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For immunoblot analysis, cell were lysed in 1× Laemmli buffer and then separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE gels). Proteins were then transferred to Immobilon®-P PVDF membrane (EMD Millipore, Billerica, MA, USA), using wet transfer. Following transfer, membranes were blocked in 5% milk in TBS plus 1% Tween® 20. Membranes were incubated with primary and secondary antibodies in 5% milk for 30 minutes. Following each antibody incubation, membranes were washed three times with tris-buffered saline (TBS) plus 1% Tween 20. Quantitation was done using densitometry on Photoshop CS6.
3
Tissue Lysate Preparation and Western Blot Analysis
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For the preparation of tissue lysates, hearts from mice were dissected and washed with ice-cold PBS, then homogenized with RIPA buffer (Beyotime Institute of Biotechnology) containing 150 mmol/l NaCl as previously described (22 (link)). Cell lysates were prepared for western blotting as previously described (23 (link)). Protein concentrations were determined using a BCA protein assay kit (Beyotime Institute of Biotechnology). After adjustment of protein concentration, the lysates were boiled in SDS loading buffer for 5 min then resolved by 10% SDS-PAGE and 20 µg of protein was loaded per lane. Gels were then transferred to an Immobilon-P PVDF membrane (EMD Millipore), which was blocked by 5% non-fat dried milk in TBST (Tris Buffer Saline with 0.1% Tween 20) buffer for 2 h at room temperature and incubated with primary antibodies against mouse NPRA (cat. no. LS-C264634; 1:500; LifeSpan BioSciences, Inc.) and GAPDH (cat. no. 2118, 1:1,000; Cell Signaling Technology, Inc.) in TBS + 0.1% Tween-20 (TBST) and 5% non-fat dry milk overnight at 4°C. Membranes were subsequently washed in 5% milk/TBST and incubated with HRP-conjugated secondary antibody for 2 h at room temperature. Protein bands were visualised using a chemiluminescence detection kit (EMD Millipore).
4
Vimentin and Actin Expression Analysis
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Cells were lysed in 1% Triton-X100, 20 mM HEPES-NaOH (pH 7.5), 500 mM NaCl, and protease/phosphatase inhibitor cocktail (Nacalai Tesque Inc., Kyoto, Japan), and the soluble fractions were collected by centrifugation at 20000 × g for 15 min at 4°C. Fractions were analyzed by SDS-PAGE, and proteins in the gel were transferred to an Immobilon-P PVDF membrane (EMD Millipore, Billerica, MA, USA). Proteins were detected with a mouse monoclonal antivimentin antibody (V9, Sigma) and anti-β-actin antibody (Sigma). Detection was performed using a C-DiGit® Blot Scanner (LI-COR Inc., Lincoln, NE, USA). Blotting data were processed using Image Studio Lite software (LI-COR).
5
Macrophage Activation and Glucocorticoid Receptor Analysis
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THP-1 monocytes were seeded in a 6-well plate with a cell density of 2,100,000 cells/mL and differentiated into macrophages by adding 200 nM of PMA to the cell culture medium and culturing them for 72 h. After differentiation, cells were exposed to 50% DPBS, 10% (v/v) AGEs, and 3 ng/mL LPS for 24 h. After incubation, cells were lysed with a Triton buffer consisting of 150 mM sodium chloride, 0.1% Triton-X, and 50 mM Tris pH 8, and the protein content was assessed with the BCA Assay (Thermo Scientific, Waltham, USA) according to the manufacturer’s protocol.
A sample of 10 µg with reducing laemmli buffer was loaded onto a 10% mini protean TGX precast gel (Bio-rad, Hercules, CA, Verenigde Staten) and run in a Bio-rad cell. Protein was transferred to an Immobilon-P PVDF Membrane (Merck, Darmstadt, Germany). Membranes were analyzed using an Amersham Imager 600 (GE Healthcare, Chicago, IL, USA). The primary antibodies used were Glucocorticoid Receptor (D6H2L), Phospho-Glucocorticoid Receptor (Ser211), and β-actin (13E5) from Cell Signaling Technology (Danvers, MA, USA) and Anti-phospho-Glucocorticoid receptor Ser226 from Millipore (Temecula, USA). The secondary antibody used was Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology).
A sample of 10 µg with reducing laemmli buffer was loaded onto a 10% mini protean TGX precast gel (Bio-rad, Hercules, CA, Verenigde Staten) and run in a Bio-rad cell. Protein was transferred to an Immobilon-P PVDF Membrane (Merck, Darmstadt, Germany). Membranes were analyzed using an Amersham Imager 600 (GE Healthcare, Chicago, IL, USA). The primary antibodies used were Glucocorticoid Receptor (D6H2L), Phospho-Glucocorticoid Receptor (Ser211), and β-actin (13E5) from Cell Signaling Technology (Danvers, MA, USA) and Anti-phospho-Glucocorticoid receptor Ser226 from Millipore (Temecula, USA). The secondary antibody used was Anti-rabbit IgG, HRP-linked Antibody (Cell Signaling Technology).
6
Wheat Shoot Microsomal Protein Extraction and Western Blot Analysis
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Wheat shoots were ground in 5 vol of an extraction buffer (100 mM Tris-HCl [pH 7.5], 25% (w/v) sucrose, 5% (v/v) glycerol, 10 mM EDTA, 5 mM KCl, and 1 mM DTT) and the supernatant was recovered from centrifugation at 15,000 g for 15 min. Microsomal fraction was obtained as the pellet of a further centrifugation at 150,000 g for 60 min. After resuspension of the pellet in the extraction buffer, the protein was subjected to SDS-PAGE and then transferred to Immobilon-P PVDF membrane (Merck Millipore). TaBx5 was probed using a polyclonal antibody raised against a TaBx5 fragment as a primary antibody and anti-rabbit IgG HRP conjugate (Promega, Madison, WI, USA) as a secondary antibody followed by detection with ECL Prime Western Blotting Detection Reagent (GE Healthcare UK Ltd., Buckinghamshire, UK) as a substrate.
7
Immunoblotting Protocol for Protein Detection
8
Western Blotting for CD200 Expression
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Cells were lysed in whole-cell extraction buffer (20 mM HEPES-NaOH, 0.5% NP-40, 15% glycerol) containing a complete protease inhibitor cocktail (Roche, Basel, Switzerland). The proteins were separated in a 12% SDS-polyacrylamide gel and transferred to an Immobilon-P PVDF membrane (Merck Millipore). Blots were incubated for 1 h at RT with a mouse monoclonal antibody recognizing human CD200 (Proteintech, Rosemont, IL, USA) or a mouse polyclonal antibody recognizing human β actin (Santa Cruz). After washing in TBS-T, the membranes were incubated for 1 h at RT with horseradish peroxidase-linked anti-mouse IgG (GE healthcare, Chicago, IL, USA). ECL Western Blotting Detection Reagents (GE Healthcare) were used to develop the high-performance chemiluminescence film (GE Healthcare).
9
Tricine-SDS-PAGE and Western Blotting
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16% Tricine–SDS-PAGE was performed as described hitherto (Schägger, 2006). The protein sample was mixed with tricine sample buffer (Bio-Rad) at 1:1, incubated at 95°C for 5 min, and subsequently centrifuged at 10,000 rpm for 30 s before loading. Electrophoresis was carried out at constant 120 V for 90 min. For protein visualization, the gel was stained with Coomassie Brilliant Blue G-250 for 45 min and then destained by using 40% methanol and 10% acetic acid mixture for 2–3 h.
For Western blotting, the resolved proteins were electrotransferred in wet conditions onto a charged Immobilon-P PVDF membrane (Merck, Germany). Non–specific binding of antibodies in the subsequent steps was prevented by membrane blocking with 5% (w/v) skim milk in TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20), overnight at 4°C. The washed membrane was probed with mouse anti-histidine monoclonal antibodies conjugated with horseradish peroxidase (Bio-Rad, CA, United States) in TBST (1:2,500) and incubated for 1 h at room temperature. The bands were visualized using Pierce™ enhanced chemiluminescent (ECL) substrate (Thermo Scientific, MA, United States).
For Western blotting, the resolved proteins were electrotransferred in wet conditions onto a charged Immobilon-P PVDF membrane (Merck, Germany). Non–specific binding of antibodies in the subsequent steps was prevented by membrane blocking with 5% (w/v) skim milk in TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20), overnight at 4°C. The washed membrane was probed with mouse anti-histidine monoclonal antibodies conjugated with horseradish peroxidase (Bio-Rad, CA, United States) in TBST (1:2,500) and incubated for 1 h at room temperature. The bands were visualized using Pierce™ enhanced chemiluminescent (ECL) substrate (Thermo Scientific, MA, United States).
10
Western Blot for Protein Analysis
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Protein extracts were heat-denaturated (95 °C) in Laemmli buffer (50 mM Tris/HCl, 0.01% Bromophenol Blue, 1.75% 2-mercaptoethanol, 11% glycerol, 2% SDS) and separated by 10–12% SDS/PAGE electrophoresis. Proteins were transferred to Immobilon-P PVDF membrane (Merck Millipore, Burlington, MA, USA). The membranes were incubated for 1h using 5% low-fat milk solution in 1X TBS (50 mM Tris-Cl, pH 7.5, 150 mM NaCl) as a blocking buffer, and then overnight at 4 °C in the same blocking solution containing one of the following antibodies: anti-HAX1 (rabbit, Proteintech 11266-1-AP) or anti-RPL26 (rabbit, 1:5000, Abcam, ab59567). After washing (3 × 10 min in TBS), membranes were incubated for 2 h at room temperature with the adequate HRP-conjugated secondary antibody: goat anti-rabbit IgG (1:5000, Abcam, GB; cat. 97051) or goat anti-mouse IgG (1:10,000, Abcam, GB; cat. ab97023). Membranes were developed using HRP detection kit WesternBright Quantum (Advansta, San Jose, CA, USA; cat. K-12042).
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