The sample was mixed with an approximate volume of 5 × sample loading buffer [250 mM Tris–HCl (pH 6.8), 10% SDS, 50% glycerol, 0.025% bromophenol blue, 250 mM dithiothreitol (DTT); without DTT in case of non-reducing condition) and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Either the gels were stained for protein with Coomassie Brilliant Blue (Nacalai, Kyoto, Japan) or the proteins were transferred to membrane Immobilon-P membranes (0.45 µm; Merck Millipore, Tokyo, Japan) for Western blotting. Anti-IgG (H + L chain) (Human) pAb-HRP (Medical & Biological Laboratories CO., LTD, Nagoya, Japan) was used for detection by Western Lightning Plus system (PerkinElmer, Waltham, MA, USA). The target protein bands were visualized through luminescent image analyzer (LAS-4000 mini; Fujifilm, Tokyo, Japan).
Western lightning plus system
Manufactured by PerkinElmer
Sourced in United States
The Western Lightning Plus system is a chemiluminescent detection solution for Western blot analysis. It provides a sensitive and reliable method for detecting and quantifying proteins separated by gel electrophoresis and transferred to a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Criterion tgx precast midi protein gel, by Bio-Rad (2 mentions)
Laemmli loading buffer, by Bio-Rad (2 mentions)
Pierce 660 nm protein assay kit, by Thermo Fisher Scientific (2 mentions)
Fusion fx system, by Vilber (2 mentions)
Las 4000 mini, by Fujifilm (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Coomassie brilliant blue, by Nacalai Tesque (1 mentions)
Mini protean tgx stain free gel, by Bio-Rad (1 mentions)
Pierce 660 nm protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Las 4000 system, by Fujifilm (1 mentions)
Pierce 660 nm protein assay, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
5 protocols using western lightning plus system
1
SDS-PAGE and Western Blotting Protein Analysis
2
Western Blot Analysis of Synaptic Proteins
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Synaptogliosome pellets were sonicated three times for 10 s at 20 Hz (Vibra cell VCX130) and boiled in Laemmli loading buffer. Protein content was measured using the Pierce 660 nm protein assay reagent (Thermo Scientific, Waltham, MA, USA). Equal amounts of proteins were separated by denaturing electrophoresis in Mini-Protean TGX stain-free gels (Biorad) and then electrotransferred to nitrocellulose membranes using the Trans-blot Turbo Transfer System (Biorad). Membranes were hybridized as described previously (Ezan et al., 2012 (link)). The antibodies used in this study are listed in the Key Resources Table . Horseradish peroxidase activity was visualized by enhanced chemiluminescence in a Western Lightning Plus system (Perkin Elmer, Waltham, MA, USA). Chemiluminescent imaging was performed on a LAS4000 system (Fujifilm, Minato-ku, Tokyo, Japan). At least four independent samples were analyzed in each experiment. The level of chemiluminescence for each antibody was normalized against stain-free signal on membranes.
3
Immunoblotting of Membrane Proteins
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MV pellets were sonicated three times for 10 s at 20 Hz (Vibra cell VCX130) in 2% SDS and heated at 56°C in Laemmli loading buffer (Biorad). The protein content was measured using the Pierce 660 nm protein assay kit (Thermo Scientific, Waltham, MA, USA). 10 µg of proteins were separated by denaturing electrophoresis on a 4-15% Criterion™ TGX™ Precast Midi Protein Gel (Biorad) and then electrotransferred to nitrocellulose membranes using the Trans-blot Turbo Transfer System (Biorad). Membranes were hybridized, as described previously (Ezan et al. 2012 ). The antibodies used in the present study are listed in the key resource table.
Horseradish peroxidase activity was visualized by enhanced chemiluminescence in a Western Lightning Plus system (Perkin Elmer, Waltham, MA, USA). Chemiluminescent imaging was performed on a FUSION FX system (Vilber, South Korea). The level of chemiluminescence for each antibody was normalized against the histone 3 staining on the membrane.
Horseradish peroxidase activity was visualized by enhanced chemiluminescence in a Western Lightning Plus system (Perkin Elmer, Waltham, MA, USA). Chemiluminescent imaging was performed on a FUSION FX system (Vilber, South Korea). The level of chemiluminescence for each antibody was normalized against the histone 3 staining on the membrane.
4
Extraction and Quantification of Brain Proteins
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Proteins were extracted from one brain hemisphere or from purified MVs in 2% SDS (500 µl or 50µl per sample, respectively) with EDTA-free Complete Protease Inhibitor (Roche), sonicated three times at 20 Hz (Vibra cell VCX130) and centrifuged for 20 min at 10,000 g at 4 °C. Supernatants were heated in Laemmli loading buffer for 5 min at 56 °C. Proteins were extracted from one brain hemisphere per sample in 500 µL SDS 2%, under the same conditions. The protein content was measured using the Pierce 660 nm protein assay (Thermo Scientific, Waltham, System (Biorad). Membranes were hybridized, as described previously [23] . The antibodies used in this study are listed in Table S11. Horseradish peroxidase activity was visualized using enhanced chemiluminescence in a Western Lightning Plus system (Perkin Elmer, Waltham, MA, USA). Chemiluminescent imaging was performed on a FUSION FX system (Vilber, South Korea). At least four independent samples were analyzed in each experiment. The level of chemiluminescence for each antibody was normalized against that of a stain-free membrane, or histone H3.
5
Western Blot Analysis of Protein Expression
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MV pellets were sonicated three times for 10 s at 20 Hz (Vibra cell VCX130) in 2% SDS and heated at 56°C in Laemmli loading buffer (Biorad). The protein content was measured using the Pierce 660 nm protein assay kit (Thermo Scientific, Waltham, MA, USA). 10 µg of proteins were separated by denaturing electrophoresis on a 4-15% Criterion™ TGX™ Precast Midi Protein Gel (Biorad) and then electrotransferred to nitrocellulose membranes using the Trans-blot Turbo Transfer System (Biorad). Membranes were hybridized, as described previously (Ezan et al., 2012) . The antibodies used in the present study are listed in Table S5. Horseradish peroxidase activity was visualized by enhanced chemiluminescence in a Western Lightning Plus system (Perkin Elmer, Waltham, MA, USA). Chemiluminescent imaging was performed on a FUSION FX system (Vilber, South Korea). The level of chemiluminescence for each antibody was normalized against the histone 3 staining on the membrane.
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