Islets were isolated as described above and homogenized in lysis buffer (Cell Signaling Technology) by sonication. Protein extracts were separated by SDS-PAGE (10% [v/v] Bis/Tris gel; Life Technologies), blotted on a nitrocellulose membrane, blocked in 4% (w/v) milk, and incubated with primary antibody (anti-hGH, ab15317, 1/1,000; anti-GAPDH, 1/15,000; clone 6C5, both from Abcam). The blot was subsequently incubated with peroxidase-conjugated secondary antibody (Dako), and proteins were detected using the Western Lightning ECL System (PerkinElmer). For hGH staining on islets from MIP-GFP and RIP-Cre mice, a similar protocol was used with adjustments: 10 μg of islet protein sample in 1× Laemmli sample buffer was resolved on 15% SDS-PAGE and transferred to polyvinylidene difluoride membranes (EMD Millipore).
Western lightning ecl system
Manufactured by PerkinElmer
Sourced in United States
The Western Lightning-ECL system is a chemiluminescent detection method used for the analysis of proteins in western blotting applications. It provides a sensitive and reliable way to detect and quantify target proteins.
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Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (2 mentions)
Immobilon p polyvinylidene difluoride pvdf membrane, by Merck Group (2 mentions)
Las 4000, by Cytiva (2 mentions)
Hrp labeled goat anti mouse igg, by LGC (2 mentions)
Peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Ab15317, by Abcam (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Clone 6c5, by Abcam (1 mentions)
Las 4000 image analyzer, by GE Healthcare (1 mentions)
Novex 6 tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Las4000 image analyser, by GE Healthcare (1 mentions)
Multi beads shocker, by Yasui Kikai (1 mentions)
Horseradish peroxidase conjugated igg, by PerkinElmer (1 mentions)
Bradford assay, by Merck Group (1 mentions)
9 protocols using western lightning ecl system
1
Western Blot Analysis of Islet Proteins
2
Fungal Protein Extraction and Western Blot
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Strains were cultured on DPY medium for 18 h. The fungal mycelia were frozen in liquid nitrogen and subsequently ground using a multi-bead shocker. After extraction, cell lysates were incubated with a buffer containing detergent NP-40 [50 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM/mL PMSF, 1× Protease Inhibitor Cocktail (Sigma, St. Louis, MO, USA: P8215), 1% NP-40] to solubilize the membrane proteins. Mycelial extract solubilized with the buffer was incubated in ice for 20 min. After centrifugation at 1000×g for 10 min, the supernatant was collected. Cell lysates were separated using SDS-PAGE. The proteins were transferred to Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore Sigma, Burlington, MA, USA) using a semidry blotting system (Nihon Eido, Tokyo, Japan). To detect EGFP, Living Colors® A.V. (anti-GFP) monoclonal antibody (1: 2000 dilution, cat # 632380, Clontech) and peroxidase-labeled anti-mouse IgG (H + L) antibody (1: 2000 dilution, cat # PI-2000, Vector Laboratories, Newark, CA, USA) were used as primary and secondary antibodies, respectively. Chemiluminescence was detected using a Western Lightning-ECL system (PerkinElmer, Waltham, MA, USA) and an LAS-4000 image analyzer (GE Healthcare, Buckinghamshire, UK).
3
SDS-PAGE, Western Blotting, and Native PAGE
4
Western Blot Analysis of Ago2 Pulldown
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For SDS-PAGE western blot the samples obtained in the Ago2 pull down assays were mixed with an appropriate amount of loading dye (25 mM Tris, 192 mM Glycine, 20% v/v glycerol, 4% m/v SDS, 0.1% v/v bromophenol blue in milli-Q water). The samples were denatured at 95°C for 5 minutes before electrophoresis on a Novex 6% Tris-glycine gel (Invitrogen). The samples were transferred to an Immobilin-P Transfer Membrane at 25V for 50 minutes. The membrane was then blocked for 60 minutes in 5% milk powder in PBS. The membrane was washed with 0.1% milk-PBS and incubated overnight at 4°C in 10 ml 0.1% milk-PBS with anti-FLAG primary antibody (1:5000) or alpha-actin (1:5000) with slow orbital mixing. After overnight incubation, the membrane was washed 3–4 times for 5 minutes with 0.1% milk-PBS and then incubated for at least one hour with slow orbital mixing in 10 ml 0.1% milk-PBS with goat anti-mouse secondary antibody (1:5000). The membrane was washed 2 times for 5 minutes in PBS-Tween, 2 times for 5 minutes in PBS and 2 times for 5 minutes in deionized H2O. The Western Lightning ECL system (PerkinElmer Life Sciences) was used for luminometric detection with the LAS4000.
5
Western Blot Protein Quantification
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Protein concentrations were determined using the Pierce BCA Protein assay kit (Thermo Fisher Scientific). Proteins were denatured, separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blocked with 5% nonfat dry milk in PBS with 0.2% Triton X-100. Membranes were labeled overnight with primary antibody at 4°C. Subsequently, the blot was incubated with horseradish peroxidase-conjugated secondary antibodies (Dako) and proteins were detected with the Western Lightning ECL System (Perkin Elmer). Images were quantified using ImageJ software (Schneider et al., 2012 (link)).
6
SDS-PAGE and Western Blotting for Protein Detection
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SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were performed as previously described [40] (link). Env was detected using MAb PA1 (0.2 µg/ml) and HRP-labeled goat anti-mouse IgG (1∶5000; Kirkegaard & Perry Laboratories, Maryland, USA) followed by the Western Lightning ECL system (PerkinElmer, Groningen, the Netherlands) and His-tagged cytokines (hAPRIL, rAPRIL and rCD40L) were detected using anti-His MAb (0.25 µg/ml) followed by HRP-labeled goat anti-mouse IgG (1∶5000; Kirkegaard & Perry Laboratories).
7
Detecting Protein-Protein Interactions via BiFC
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Mycelia of the strain NgACgB1, expressing nEGFP-LZA and cEGFP-LZB, were inoculated into 20 ml DPY liquid medium and incubated at 30 °C for 18 h. The frozen mycelia (250 mg) were homogenised with a Multi-beads Shocker (Yasui Kikai, Osaka, Japan) and then suspended in 1 ml extraction buffer (250 mM sucrose, 50 mM Tris-HCl (pH 8.0), and 1 mM phenylmethylsulfonyl fluoride [PMSF]) supplemented with 1% protease inhibitor cocktail (Sigma, St. Louis, MO, USA). The lysate was centrifuged at 1,000 × g for 10 min, and the supernatant was analysed by SDS-PAGE on 15% polyacrylamide gel. For immunoblotting, proteins in the gel were transferred onto Immobilon-P polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA) via a semidry blotting system (Nihon Eido, Tokyo, Japan). For detection of nEGFP and cEGFP, anti-GFP polyclonal antibody (Code No. 598, MBL, Nagoya, Japan) was used. Chemiluminescence was detected using a Western Lightning-ECL system (PerkinElmer, Waltham, MA) and an LAS-4000 image analyser (GE Healthcare, Buckinghamshire, UK).
8
Immunoprecipitation and Western Blotting
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Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and then lysed with 1% Nonidet P-40 lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 20 mg/ml leupeptin) on ice for at least 20 min. Next, lysates were collected and clarified by centrifugation at maximum speed for 30 min at 4°C. Immunoprecipitations were carried out by incubating cell lysates with antibodies, as indicated, for 4 hr at 4°C, followed by incubation for an additional 3 hr with protein A-sepharose 4B beads. Then, after washing three times with lysis buffer, immune complexes were resolved using SDS-PAGE. Finally, Western blotting was performed using horseradish peroxidase-conjugated IgG as a secondary antibody and the Western Lightning-ECL system (PerkinElmer Inc., Waltham, MA) for detection. All experiments were conducted at least three independent times.
9
Protein Co-Immunoprecipitation and Western Blotting
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Proteins were extracted and subjected to co-immunoprecipitation and/or Western blotting as described 10 . Briefly, cells were washed twice with ice-cold PBS and then lysed with 1% Nonidet P-40 lysis buffer (20 mM Tris, pH 8.0, 137 mM NaCl, 1% Nonidet P-40, 10% glycerol, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 20 mg/ml leupeptin), harvested by scraping, and on the ice for 30 min. Cell lysates were collected and clarified by centrifugation for 25 min at 4 °C, and total protein concentration was determined using the Bradford Assay according to the manufacturer's instructions (Sigma-Aldrich, St Louis, MO). Immunoprecipitations were carried out by incubating cell lysates with antibodies as indicated for 12 h at 4 °C, followed by incubation for 4 h with protein A-Sepharose 4B beads. After washing five times with lysis buffer, immune complexes were resolved using SDS-PAGE. Western blotting was proceeded, and then incubated with indicated primary antibodies. The membranes were incubated with horseradish peroxidase-conjugated IgG as a secondary antibody and the Western Lightning ® -ECL system (PerkinElmer Inc., Waltham, MA) for detection.
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