ProteoExtract Transmembrane Protein Extraction Kit (Calbiochem) was used for protein isolation according to the manufactures protocol ‘Extraction of Membrane Proteins from Adherent Tissue Culture Cells’. Protein concentration was determined by Pierce BCA assay and 10 µg protein per lane was loaded on 12.5% polyacrylamid gels. Subsequent to SDS gel electrophoresis, proteins were blotted onto polyscreen PVDF transfer membranes (Perkin Elmer, Waltham, MA). Blots were blocked in 5% BSA before overnight incubation with primary antibodies (mouse-anti claudin-4 and -5 or rabbit-anti caudin-3, -7 and -18, all Zymed/Invitrogen, 1∶1000). Peroxidase-conjugated AffiniPure F(ab′)2 fragment goat anti-mouse/−rabbit (1∶10000, Jackson ImmunoResearch) were used as secondary antibodies and Lumi-LightPLUS Western Blotting Kit (Roche, Grenzach-Wyhlen, Germany) was used for visualizationin a Fusion FX 7 image acquisition system (Vilber Lourmat, Eberhardzell, Germany).
Lumi lightplus western blotting kit
Manufactured by Roche
Sourced in Switzerland, Germany
The Lumi-LightPLUS Western Blotting Kit is a laboratory equipment product designed for the detection and visualization of proteins in Western blot analysis. It provides the necessary reagents and materials for performing the chemiluminescent detection step of the Western blotting procedure.
Automatically generated - may contain errors
Lab products found in correlation
Fusion fx7, by Vilber (2 mentions)
Las 4000 image analyzer, by Fujifilm (2 mentions)
Polyscreen pvdf transfer membrane, by PerkinElmer (1 mentions)
Amersham hyperfilm ecl, by GE Healthcare (1 mentions)
Protease inhibitor pmsf, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Protein lysis buffer, by Beyotime (1 mentions)
Chemiluminescence imaging system, by Clinx (1 mentions)
Loading buffer, by Transgene (1 mentions)
Plant total protein extraction kit, by Sangon (1 mentions)
Myc tag, by Cell Signaling Technology (1 mentions)
Las 3000, by Fujifilm (1 mentions)
Pvdf microporous membrane, by Merck Group (1 mentions)
Las 1000, by Fujifilm (1 mentions)
Las 4000, by Fujifilm (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti occludin, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor mixture, by Roche (1 mentions)
Las 1000 imaging system, by Fujifilm (1 mentions)
13 protocols using lumi lightplus western blotting kit
1
Transmembrane Protein Extraction and Analysis
2
Western Blot Protein Detection Protocol
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Total cell protein extracts were prepared using RIPA buffer as previously described [26 (link)]. All protein samples were electrophoresed in a SDS-polyacrylamide gel and transferred onto PVDF membranes. PVDF membranes were blocked for 2 hours with 5% non-fat milk or 5% BSA in TBS containing 0.1% Tween-20, and then incubated overnight at 4°C with primary antibodies. Membranes were washed three times with TBS-Tween 0.05% and incubated with HRP-conjugated anti-rabbit IgG or anti-mouse IgG secondary antibodies for 1 hour at room temperature in 5% non-fat dry milk in TBS-Tween. Immunoreactive bands were developed with Lumi-lightPLUS Western Blotting Kit (Roche, Molecular Biochemicals) and visualized using X-ray films (Amersham Hyperfilm ECL, GE Healthcare, Buckinghamshire, UK).
3
Western Blot Analysis of Transfected Cells
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At 48 h post-transfection (p.t.), cells were washed twice with PBS and lysed with 100 µl protein lysis buffer (Beyotime, Shanghai, China) containing 1% PMSF protease inhibitors (Beyotime). After 30 min on ice, lysates were centrifuged at 12 000×g for 15 min and the supernatants were collected. The concentration of proteins in the supernatant was then measured using a BCA Protein Assay Kit (Beyotime). Approximately 25 µg of total proteins were resolved by 12% SDS-PAGE and analyzed by immunoblotting by using mouse monoclonal α-GP64 antibody (1∶2000, Abcam), mouse monoclonal α-EGFP antibody (1∶2000, Abcam) and mouse monoclonal α-tubulin antibody (1∶2000, Beyotime). The horseradish peroxidase-conjugated goat α-mouse IgG (1∶20000, Beyotime) was used as the secondary antibody. Protein bands were visualized using the Lumi-Light PLUS Western Blotting Kit (Roche) and a Chemiluminescence Imaging System (Clinx Science Instruments, Shanghai, China).
4
Protein Extraction and Western Blot Analysis
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Total soluble protein was extracted from the seeds at 30–40 days after pegging (DAP) of control FH1 and the transgenic T3 generation lines using a plant total protein extraction kit (Sangon Biotech Co., Ltd., Shanghai, China). Total protein extracts of over 40–50 μg for each sample were boiled for 5 min in 6× loading buffer (Transgen Biotech, Beijing, China), separated by 10% sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a 0.45-μm polyvinylidene difluoride (Roche Applied Science, Mannheim, Germany) membrane by the wet electrotransfer method (Tovey and Baldo, 1987 (link)). After transfer, blotting efficiency was checked by reversibly staining the transferred proteins with Ponceau S solution. Using mouse monoclonal antibody Myc-Tag (9B11; Cell Signaling Technology, Inc.) as the primary antibody, western blotting was carried out in accordance with the instructions of the Lumi-LightPLUS Western Blotting Kit (Roche Applied Science).
5
Western Blot Analysis of Signaling Proteins
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Whole-cell lysates and whole-skin lysates were subjected to SDS-PAGE, and the proteins that migrated were electrically transferred to a polyvinylidene fluoride (PVDF) microporous membrane (Life Technologies, MA, USA). The membrane was incubated at 4°C for 18 h with antibodies against the following proteins: p-Akt, Akt, p-p38 MAPK, p38 MAPK, p-CREB, CREB, p-PI3K, PI3K (1:1000), and GAPDH (1:2000). After incubation, anti-mouse HRP-linked secondary antibody (1:2000, for GAPDH) or anti-rabbit IgG HRP (1:2000, for p-Akt, Akt, p-p38 MAPK, p38 MAPK, p-CREB, CREB, p-PI3K, and PI3K) was added, and the membrane was incubated for 30 min at room temperature. The chemiluminescence of antigenic proteins on the membrane was monitored using the Lumi-LightPLUS western blotting kit (Roche Diagnostics Co., Basel, Switzerland), and the images were analyzed using an image analyzer (LAS-3000; Fujifilm Co., Tokyo, Japan).
6
Western Blot Analysis of Cellular Signaling
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Western blot analysis was performed using the method described previously21 (link). Whole-cell lysates and plasma membrane fractions were subjected to SDS-PAGE, and the proteins that had migrated were electrically transferred to a microporous polyvinylidene fluoride membrane (Millipore Corporation, MA, USA) for western blotting21 (link). The membrane was incubated at 4 °C for 18 h with antibodies against the following proteins: p-Akt, Akt, p-AMPK, AMPK, p-ACC, ACC (all at 1:1000), and β-actin (1:2000)21 (link). After incubation, anti-mouse (1:2000 for β-actin) or anti-rabbit IgG horseradish peroxidase conjugate (1:2000 for p-Akt, Akt, p-AMPK, AMPK, p-ACC, and ACC) was added, and the membrane was incubated for 30 min at RT. The antigenic proteins on the membrane were visualized via chemiluminescence using a Lumi-LightPLUS Western Blotting Kit (Roche Diagnostics Co., Basel, Switzerland), and the images were evaluated using an Image Analyzer LAS-4000 (Fujifilm Co. Tokyo, Japan)21 (link).
7
Western Blot Analysis of Signaling Proteins
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Whole-cell lysates and plasma membrane fractions were subjected to SDS-PAGE, and the proteins that had migrated were electrically transferred to a polyvinylidene fluoride (PVDF) microporous membrane (Millipore Corporation, MA, USA) for Western blotting. The membrane was incubated at 4°C for 18 h with antibodies against the following proteins: p-Akt, Akt, p-AMPKα, AMPKα, p-ACC, ACC, p-LKB1, LKB1, p-PDK1, PDK1, GLUT4 (1∶1000), E-cadherin (1∶200), and β-actin (1∶2000). After the incubation, anti-mouse IgG horseradish peroxidase-conjugate (HRP, 1∶2000, for β-actin) or anti-rabbit IgG HRP (1∶2000, for p-Akt, Akt, p-AMPKα, AMPKα, p-ACC, ACC, p-LKB1, LKB1, p-PDK1, PDK1, and GLUT4) was added; and the membrane was incubated for 30 min at room temperature. The antigenic proteins on the membrane were visualized by chemiluminescence by use of a Lumi-LightPLUS Western blotting kit (Roche Diagnostics Co., Basel, Switzerland), and the images were evaluated by using an Image Analyzer LAS-4000 (Fujifilm Co. Tokyo, Japan).
8
Western Blot Analysis of Tight Junctions
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Western blot was performed as described previously (Markov et al. 2014) . Briefly, tissues were homogenized in Tris-buffer containing (in mM) Tris (20), MgCl 2 (5), EDTA (1), EGTA (0.3) and protease inhibitors (Complete, Boehringer, Mannheim, Germany), and protein contents were determined using BCA protein assay reagent (Pierce, Rockford, IL, USA) and quantified with a plate reader (Tecan, Groedig, Austria). Samples were mixed with SDS buffer (Laemmli), loaded on a 12.5% SDS polyacrylamide gel and electrophoresed. Proteins were detected by immunoblotting, employing primary antibodies raised against occludin or claudin -1, claudin-2, claudin-3, claudin-4, claudin-5, claudin-7 andclaudin-8 (Life Technologies 6 , USA). Peroxidase-conjugated goat antirabbit IgG or goat anti-mouse IgG antibodies and the chemiluminescence detection system Lumi-LightPLUS Western blotting kit (Roche, Mannheim, Germany) were used to detect bound antibodies. Signals were visualized by luminescence imaging (LAS-1000; Fujifilm, Tokyo, Japan), and densitometry was performed using AIDA and Biorad Quantity One software, taking into account beta-actin bands of self-same immunoblots, respectively. Data were corrected, employing a correction factor in accordance with morphometric analysis.
9
Western Blot Analysis of Nitric Oxide Synthases
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Homogenates were assayed for nNOS and iNOS protein levels by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot procedures as previously described (Gądek-Michalska et al. 2015 (link)). Blots containing proteins were exposed to the following antibodies: primary rabbit anti-nNOS (1:400, sc-648) and anti-iNOS (1:400, sc-650), polyclonal antibodies, primary mouse anti-β-actin (1:400, sc-47778), monoclonal antibody followed by goat anti-rabbit (1:10000, sc-2004), and goat anti-mouse (1:4000, sc-648) horseradish peroxidase–conjugated secondary antibodies, all of which were provided by Santa Cruz Biotechnology, Dallas, TX, USA. Proteins were visualized by enhanced chemiluminescence (Lumi-LightPlus Western Blotting Kit, Roche Diagnostics, Switzerland). Immunoblots were subsequently evaluated using a luminescent image analyzer (LAS-4000, Fujifilm, Japan). The optical densities of the appropriate bands were quantified by densitometry using Image Gauge V4.0 software (Fujifilm, Japan) and normalized to β-actin levels. All the values are expressed as a percentage of controls.
10
Western Blot Analysis of OsbZIP23 Protein
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The total protein was isolated from leaves of transgenic and NT rice plants and western blot analysis was carried out taking 40 μg of total protein following the previously reported method [37 (link)]. Affinity purified polyclonal antibody raised against OsbZIP23 protein in rabbit (catalog no- E580041-A-SE, EnoGene Biotech, USA) was used as primary antibody (1: 1,000 dilution) and monoclonal plant anti-actin antibody (Sigma, catalog no.-A0480) was used as loading control (1:500 dilution). Immuno-detection was carried out with the Lumi-LightPLUS western blotting kit (Roche Molecular Biochemicals), following manufacturer’s instruction.
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