The structural integrity of the Ad5 fiber proteins was assessed by Western blotting. An amount of 5×109 vp/virus stock were run on ready-made 10% NuPAGE polyacrylamide gels (Invitrogen, Paisley, UK) by SDS-PAGE and transferred to Hybond-P nitrocellulose membrane (GE Healthcare Life Sciences, Little Chalfont, UK) by semidry blotting. Nitrocellulose membranes were treated with 5 ml of Pierce Miser antibody extender (Thermo Scientific) for 10 min and washing 7 times with distilled water. They were then blocked in 5% milk in Tris-buffered saline containing 0.05% TWEEN-20 and 0.05% Triton X-100 (TBS-T) overnight at 4°C. The membrane was incubated in primary anti-adenovirus fiber antibody 4D2 (1:2000) at 37°C for 1 hr, washed 5 times for 5 min in TBS-T, and incubated in antimouse IgG-HRP conjugate (1:2000; Insight Biotechnology Ltd., Wembley, UK) for 1 hr at room temperature. After washing a further 5 times for 5 min in TBS-T, the membrane was incubated for 5 min in SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific), and analyzed on GelDoc autoChemi camera (Ultra-Violet Products Ltd., Cambridge, UK).
Hybond p nitrocellulose membrane
Manufactured by GE Healthcare
Sourced in United Kingdom
Hybond-P is a nitrocellulose membrane used in protein transfer and detection applications. It provides a stable matrix for the immobilization of proteins during Western blotting and other immunodetection techniques.
Automatically generated - may contain errors
Lab products found in correlation
Nupage polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Pierce miser antibody extender, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor cocktails 2 and 3, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti rabbit igg antibody, by GE Healthcare (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Horseradish peroxidase hrp linked anti mouse igg, by GE Healthcare (1 mentions)
Gal4 ad, by Takara Bio (1 mentions)
6 protocols using hybond p nitrocellulose membrane
1
Ad5 Fiber Protein Structural Integrity
2
Intracellular OPTN Quantification by Western Blot
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Western blotting was used to determine intracellular OPTN levels. Cells were lysed in Laemmli sample buffer (0·06 m Tris–HCl pH 6·8, 2% SDS, 10% glycerol, 5% β2-mercaptoethanol (VWR, Leicestershire UK), 0·04% [weight/volume (w/v)] bromophenol blue, Complete protease inhibitor cocktail tablet (Roche Diagnostics GmbH, Mannheim, Germany), and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich). Proteins were separated by SDS–PAGE and transferred onto Hybond P nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) with a semi-dry transfer system (Trans-Blot SD semi-dry transfer cell; Bio-Rad, Hemel Hempstead, UK) in transfer buffer [200 mm glycine, 0·1% SDS (w/v), 10% methanol (v/v), 25 mm Tris–HCl pH 8·8]. Membranes were incubated overnight at 4° with primary antibodies directed against OPTN (HPA003360; Sigma-Aldrich, 1 : 1000) or β-actin (Sigma-Aldrich; 1 : 1000) and then with horseradish peroxidase-conjugated anti-rabbit IgG antibody (GE Healthcare, Amersham, UK, 1 : 2000) for 1 hr at room temperature, and developed using the Enhanced Chemiluminescence method (ECL PLUS; GE Healthcare). Densitometry analysis was performed using imagej software (National Institutes of Health, Bethesda, MA), with band densities normalized to β-actin.
3
Western Blot Analysis of FLAG-Tagged Proteins
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Ten micrograms of total cellular protein was separated by 10% SDS-PAGE and transferred to a Hybond P nitrocellulose membrane (GE Healthcare). The membrane was blocked with 5% skim milk in TBS, incubated with the M2 anti-FLAG mouse monoclonal antibody in TBS, washed with TBS, and incubated with horseradish peroxidase (HRP)-linked anti-mouse IgG (GE Healthcare) in TBS. After being washed, the membranes were immersed in the peroxidase stain kit solution (Nacalai Tesque Inc.).
4
Immunoblotting Analysis of Recombinant Proteins
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Proteins from agroinfiltrated N. benthamiana leaves and co-transformed yeast cells were extracted as described [25 (link)]. Proteins in whole-cell lysates were subjected to sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (12% or 15% polyacrylamide gels) and transferred to a Hybond-P nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) by electroblotting. Polyclonal anti-HCpro (courtesy of F. Rabenstein, Julius Kühn-Institut, Quedlinburg, Germany) or polyclonal anti-eIF4E (courtesy of K. Browning, University of Texas, Austin, TX, USA) was used for plant samples, and a monoclonal antibody against the GAL4 AD or GAL4 DNA BD (1:50,000; Clontech, Mountain View, CA, USA) was used for yeast samples. Bound polyclonal antibodies were detected with horseradish peroxidase–conjugated anti-rabbit serum (1:250,000; GE Healthcare, Buckinghamshire, UK), and monoclonal antibodies were detected with horseradish peroxidase-conjugated anti-mouse serum (1:200,000; GE Healthcare). Detection was carried out with the Super Signal West Femto Chemiluminescent Substrate for detection of horseradish peroxidase (Thermo Scientific, Rockford, IL, USA) and visualized by exposure to X-ray film. Equal loading was controlled by Coomassie staining.
5
HAdV-C5 Fibre Knob Protein Structural Integrity
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The structural integrity of the HAdV-C5 fibre knob proteins incorporating peptide insertions was assessed by Western blotting. A total of 1 × 1010 vp/virus stock were run on pre-made 10% NuPAGE polyacrylamide gels (Invitrogen, Paisley, UK) by SDS-PAGE and transferred to Hybond-P nitrocellulose membrane (GE Healthcare Life Sciences, Little Chalfont, UK) by semidry blotting. Nitrocellulose membranes were treated with 5 mL of Pierce Miser antibody extender (Thermo Scientific) for 10 min and washed seven times with distilled water. Membranes were blocked in 5% milk in Tris-buffered saline containing 0.05% TWEEN-20 and 0.05% Triton X-100 (TBS-T) overnight at 4 °C. The membrane was incubated in primary anti-adenovirus fibre antibody 4D2 (Abcam, ab3233) (1:2000) at 37 °C for 1 h, washed five times for 5 min in TBS-T and incubated in anti-mouse IgG-HRP conjugate (1:2000; Insight Biotechnology Ltd., Wembley, UK) for 1 h at room temperature. After washing a further five times for 5 min in TBS-T, the membrane was incubated for a maximum of 10 min in Super Signal West Pico Chemiluminescent substrate (Thermo Scientific) and analysed on GelDoc autoChemi camera (Ultra-Violet Products Ltd., Cambridge, UK).
6
Western Blot Protein Analysis Protocol
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The proteins in the total cell lysates were extracted using 0.7% CHAPS Buffer (20 mM Tris [pH 7.5], 120 mM NaCl, 1 mM EDTA, 5 mM EGTA, 50 mM β-glycerophosphate, and 50 mM NaF) supplemented with 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin.
The proteins were separated using 8% or 14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated proteins were electrotransferred to a Hybond-P nitrocellulose membrane (GE Healthcare Ltd, Little Chalfont UK). Moreover, the membrane was blocked in wash buffer (10 mM Tris [pH 7.5], 150 mM NaCl, and 0.05% Tween-20) and supplemented with 2% skim milk for 30 minutes. The membrane was washed with specific primary antibodies diluted buffer and subsequently incubated with HRP-conjugated secondary antibodies for 1 h. Antibody-bound proteins were visualized using an enhanced luminol-based chemiluminescent method, and the protein bands were imaged using ChemiStage 16-CC (KURABO Industries Ltd., Osaka, Japan). The intensity of protein bands was measured using ImageJ (NIH) [21] . Where indicated, some membranes were stripped and probed with a different antibody.
The proteins were separated using 8% or 14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the separated proteins were electrotransferred to a Hybond-P nitrocellulose membrane (GE Healthcare Ltd, Little Chalfont UK). Moreover, the membrane was blocked in wash buffer (10 mM Tris [pH 7.5], 150 mM NaCl, and 0.05% Tween-20) and supplemented with 2% skim milk for 30 minutes. The membrane was washed with specific primary antibodies diluted buffer and subsequently incubated with HRP-conjugated secondary antibodies for 1 h. Antibody-bound proteins were visualized using an enhanced luminol-based chemiluminescent method, and the protein bands were imaged using ChemiStage 16-CC (KURABO Industries Ltd., Osaka, Japan). The intensity of protein bands was measured using ImageJ (NIH) [21] . Where indicated, some membranes were stripped and probed with a different antibody.
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