Purified HSV-2 lysate (Advance Biotechnologies, Columbia, MD) was resolved in 4 to 12% bis-Tris precast gradient SDS-polyacrylamide gels (Life Technologies) and blotted onto a polyvinylidene difluoride membrane using an iBlot gel transfer system (Life Technologies). Membranes were blocked with 5% bovine serum albumin (Sigma) overnight. The blocked membranes were incubated overnight at 4°C with 1 μg/ml GRFT in PBS or with mouse anti-gD MAb DL11 diluted 1:500 in 5% nonfat instant dry milk, PBS, and 0.1% Tween. The membranes were washed and incubated with rabbit polyclonal anti-GRFT (Pacific Immunology, Ramona, CA) or anti-mouse immunoglobulin conjugated with horseradish peroxidase (HRP) (Promega, Madison, WI). After the required washing steps, the membrane used to detect GRFT binding was incubated with anti-rabbit immunoglobulin conjugated with HRP and washed again, and both membranes were processed using an enhanced chemiluminescence Western blotting detection system (Thermo Scientific).
Enhanced chemiluminescence western blotting detection system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Enhanced chemiluminescence western blotting detection system is a lab equipment product that allows for the detection and visualization of proteins in western blot analysis. It utilizes a chemiluminescent substrate to generate a luminescent signal proportional to the amount of target protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor mix, by Roche (2 mentions)
Iblot gel transfer system, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti actin antibody, by Cell Signaling Technology (1 mentions)
Anti pten antibody, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by ZSGB-BIO (1 mentions)
5 protocols using enhanced chemiluminescence western blotting detection system
1
Virus Lysate Western Blotting Protocol
2
Protein Extraction and Western Blotting
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Protein was extracted from the cells and the tissues using a lysis buffer (50 mM Tris, 150 mM NaCl, 10 mM EDTA) supplemented with protease inhibitors (1 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml trypsin inhibitor) and 1% NP-40. The concentration of the protein was estimated by bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, MA). Protein (20 µg) was subjected to SDS-polyacrylamide gel electrophoresis and transferred onto the nitrocellulose membrane. The membrane was then blocked for 1 h with 3% bovine serum albumin (fatty acid free) and incubated with the primary antibodies overnight at 4 °C. Then, the blots were washed and incubated with secondary antibodies for 90 min at room temperature (RT). Blottings were again washed and the proteins were visualized using enhanced chemiluminescence western blotting detection system (Thermo Fisher Scientific, Waltham, MA). Antibody sources and dilutions used are listed in Supplementary Table 2 . All blots or gels were derived from the same experiment and were processed in parallel.
3
Protein Extraction and Western Blot Analysis
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After 6 Gy of irradiation, protein extraction from 1 × 106 transfected cells was performed with lysis buffer (20 mM Tris‐hydrochloride pH 7.4, 150 mM sodium chloride, 1% Triton X‐100, 0.1 mM ethylenediaminetetraacetic acid, 1 mM ethylene glycol tetraacetic acid, 2 mM sodium orthovanadate, 2 mM sodium fluoride, and Complete Protease Inhibitor Mix [Roche Applied Science, Mannheim, Germany]). Protein concentration was measured by BCA protein assay (Pierce, Thermo Scientific, Rockford, IL, USA) and samples were resolved on a 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis gel, followed by transfer to nitrocellulose membranes. Membranes were blocked in 5% non‐fat dry milk in Tris Buffer saline Tween‐20 at room temperature and probed with primary antibodies (anti‐PTEN antibody [Santa Cruz Biotechnology, Paso Robles, CA, USA] and anti‐ß‐actin antibody [Cell Signaling, Danvers, MA, USA]) overnight at 4°C and with secondary antibody for two hours at room temperature. Antibody complexes were visualized using an enhanced chemiluminescence‐Western blotting detection system (Thermo Scientific).
4
Western Blot Analysis of Phospho-Akt
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Cells were lysed in lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1 mM EDTA, 1 mM EGTA, 2 mM sodium orthovanadate, 2 mM NaF, and Complete TM Protease Inhibitor Mix [Roche Applied Science, Mannheim, Germany]) for 20 min on ice and cleared by centrifugation at 12,000 rpm and 4°C. Proteins were resolved on a 10% SDS PAGE gel, transferred onto nitrocellulose membranes, and blocked with 5% nonfat dry milk in TBST (10 mM Tris-HCl pH 7.5, 100 mM NaCl, and 0.05% Tween 20), followed by incubation with a primary antibody [total and anti-phosphorylated-Akt (Ser473) antibody (Cell Signaling Biotechnology, Beverly, MA, USA)]. Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibody. Antibody complexes were visualized using an enhanced chemiluminescence-Western blotting detection system (Thermo Fisher Scientific, Inc., Rockford, IL, USA).
5
NF-κB, p38MAPK, and Card9 Expression in Pancreas
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The expression of NF-κB, p38MAPK, and Card9 in the pancreas at 3, 6, and 12 h were detected by western blotting. In short, a part of the frozen samples was disposed in ice-cold lysis buffer containing protease inhibitors. Then, the protein was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Subsequently, the membranes were blocked with 5% nonfat milk overnight at 4°C. After blocking, the membranes were incubated with primary antibodies against NF-κB, p38MAPK, Card9, p-NF-κB, p-p38MAPK, and p-Card9 (ZSGB-BIO, China) overnight and with horseradish peroxidase–conjugated secondary antibodies (ZSGB-BIO) for 1 h. The bands were obtained using an enhanced chemiluminescence western blotting detection system (Thermo, USA). β-actin was used as an internal reference for relative quantification.
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