Samples were solubilized in sample buffer (3X) and boiled at 95 °C for 5 min for immunoblotting. All samples were electrophoresed on 10% (w/v) sodium dodecyl sulphate-polyacrylamide (SDS-PAGE) gels. Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes. For all immunoblot experiments, membranes were blocked for 1 h at ambient temperature in blocking buffer composed of 5% (w/v) skim milk powder in TBS. PVDF membranes were incubated overnight at 4 °C with primary antibodies. A complete list of primary antibodies and the appropriate methodology used is shown in Additional file 1 : Table S1. Membranes were then washed with TBS containing 0.05% Tween 20 and incubated for 1 h at ambient temperature with the following secondary antibodies diluted in blocking buffer: either α-mouse horseradish peroxidase (HRP)-conjugated (NA931, GE Healthcare; 1:5000), α-rabbit HRP-conjugated (NA934, GE Healthcare, 1:5000), or α-goat HRP-conjugated (sc-2433, Santa Cruz, 1:5000) antibody. Visualization of immunoblot labelling was achieved using chemiluminescence with the Western Lighting Plus ECL kit (Perkin Elmer).
Western lighting plus ecl kit
Manufactured by PerkinElmer
Sourced in United States
The Western Lighting Plus ECL kit is a chemiluminescent detection system used for the analysis of proteins in Western blot applications. It utilizes a proprietary enhanced chemiluminescent (ECL) substrate to enable sensitive detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Quantity one software, by Bio-Rad (2 mentions)
Na931, by GE Healthcare (1 mentions)
Na934, by GE Healthcare (1 mentions)
Chemidoc touch system, by Bio-Rad (1 mentions)
Precast gel, by Bio-Rad (1 mentions)
Immobilon p polyvinylidene difluoride, by Merck Group (1 mentions)
Mini protean 3 system, by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Anti gfp antibody, by Abcam (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Versadoc 5000 imaging system, by Bio-Rad (1 mentions)
Ab58023, by Abcam (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
M11217, by Thermo Fisher Scientific (1 mentions)
Donkey anti rabbit secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ma5 15256, by Thermo Fisher Scientific (1 mentions)
Apc 052, by Alomone (1 mentions)
Apc 030, by Alomone (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
10r g109a, by Biosynth (1 mentions)
6 protocols using western lighting plus ecl kit
1
Immunoblotting Technique for Protein Analysis
2
Western Blot Protein Analysis Protocol
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Protein lysates were extracted in the SDS-lysis buffer (0.5 mM EDTA, 20 mM HEPES, 2% (w/v) SDS pH 7.9). Samples were boiled to 95°C for 5 min and DNA was sheared using a 26G needle before being stored at −20°C for further processing. Equal amounts of protein lysates (10–30 μg) were loaded into 4%–15% precast gels (Bio-Rad) and ran using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in Tris-glycine-SDS running buffer. Separated proteins were then transferred onto Immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore IPVH00010) using the Mini-PROTEAN III system (Bio-Rad). After transfer, membranes were blocked with 5% milk in TBS-T blocking buffer at RT for 1 h and then incubated with primary antibodies diluted in blocking buffer at 4°C overnight. The following morning, the membranes were washed three times with TBS-T then incubated with mouse or rabbit horseradish peroxide-bound secondary antibodies followed by three washes with TBS-T. The protein was visualized using the Western Lighting Plus ECL kit (PerkinElmer). Images were acquired using ChemiDoc Touch system (Bio-Rad) and processed by Image Lab software (Bio-Rad). The primary and secondary antibodies are listed in Table S2 .
3
Immunoblotting of Rosette Leaf Proteins
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Rosette leaves from 14-day-old plants were divided into leaf blades and petioles. Each leaf part was homogenized in 50 mM sodium phosphate buffer (pH 7.0) containing 150 mM NaCl, 0.02% (w/v) NaN3, 10 mM DTT, and 0.1% (v/v) Triton X-100. An aliquot of the resulting protein extract was separated by SDS–PAGE using a 10% SDS gel and transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Billerica, MA, USA). After blocking with 3% (w/v) fat-free skimmed milk, the blotted membrane was incubated with the primary antibodies anti-BGLU18 (Ogasawara et al., 2009 (link)), anti-NAI2 (Yamada et al., 2008 (link)), anti-binding protein (BiP; Yamada et al., 2008 (link)) (each at 1:5000 dilution), and anti-GFP antibody (Abcam, Tokyo, Japan) (at 1:10 000 dilution). The membrane was incubated with anti-rabbit IgG secondary antibodies conjugated to horseradish peroxidase at 1:20 000 dilution. Chemiluminescent immunoblotting was performed using a Western Lighting Plus-ECL kit (Perkin-Elmer Life Sciences, Wellesley, MA, USA), and the signals were digitally captured on a VersaDoc 5000 imaging system using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed using ImageJ software (National Institute of Health, Bethesda, MD, USA) to determine the relative intensities of the protein bands.
4
Western Blot Protein Analysis
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Western blot analysis was performed on Mini-PROTEAN® TGX™ Gels (Bio-Rad Laboratories). Proteins were electrotransferred onto Immobilon-P PVDF membrane (millipore, pore size 0.45 mm). After blocking 1 h in Tris-HCl-buffered saline with 0.2% Tween-20 (TBST) and 5% non-fat milk (Bioshop® Canada), the membranes were incubated overnight with primary antibody in TBST containing 2% non-fat milk. The rabbit polyclonal anti-HCN2 antibody (1:500; APC-030, Alomone Labs), rabbit polyclonal anti-HCN4 antibody (1:1000; APC-052, Alomone Labs), mouse monoclonal anti-SGO1 antibody (1:1000; ab58023, Abcam), mouse monoclonal anti-GAPDH antibody (1:1000; 10R-G109a, Fitzgerald), mouse monoclonal anti-GFP antibody (1:1000; MA5-15256, Invitrogen), rat monoclonal anti-mCherry (1:1000; M11217, Invitrogen) and mouse monoclonal anti-PP2A C subunit (1:1000; SAB4200266, Sigma-Aldrich) were utilized to detect HCN2, HCN4, SGO1, GAPDH, GFP, mCherry and PP2A C proteins respectively. Detection was carried out by the use of horseradish peroxidase conjugated secondary antibodies (Donkey anti rabbit secondary antibody, 1:5000, #711035152, Jackson; Donkey anti mouse secondary antibody, 1:5000, #711035151, Jackson; Donkey anti rat secondary antibody, 1:5000, #A18745, Invitrogen), photographic film (Agfa NV) and the Western Lighting® plus ECL kit (PerkinElmer).
5
Quantitative Analysis of Muscle Protein Rbm24
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Muscle tissues and satellite cells were homogenized in lysis buffer (50 mM Tris–HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Triton-X100) supplemented with cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail and PhosSTOP Phosphatase Inhibitor Cocktail (both from Sigma-Aldrich). Protein samples were separated by SDS-PAGE and transferred to nitrocellulose sheets (GE Healthcare). Unspecific bindings were blocked using non-fat dry milk (5%) in TBST (20 mM Tris–HCl, pH 7.5, 137 mM NaCl, 0.1% Tween-20) for 1 h at room temperature. The nitrocellulose membranes were then incubated at 4 °C overnight with Rbm24 antibody (abcam, 1:200) or α-tubulin antibody (Sigma-Aldrich, 1:10,000) diluted in TBST containing bovine serum albumin (5%). After washing in TBST, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (Biorad), and protein bands were visualized using the Western Lighting Plus-ECL kit (PerkinElmer). The intensity of signals was quantified using ImageJ software (version number 1.45s).
6
Glycolytic Enzyme Activity in Cancer Cell Lines
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4T1 and Bcap37 cells (1 × 106) are seeded and allowed to attach overnight. The media are then replaced with fresh complete RPMI with 6 mM glucose, supplemented by lactic acid to a lactate level at 20 mM and pH at 6.7. Cells are collected at 0, 24, 48 and 72 hours.
For measuring the activities of individual glycolytic enzymes, whole cell lysates are prepared and the activity of each glycolytic enzyme assayed as above-described.
For immuno-blot assays, we employ a reported method53 (link). Cells are lysed in 625 mM Tris-HCl (pH 6.8), 10% SDS, 25% glycerol, 5% β-mercaptoethanol and 0.015% bromphenol blue supplemented with a protease inhibitor cocktail (Pierce, Rockford, IL), followed by sonication. Samples are stored at −80°C after heating and protein concentrations scored by the BCA protein assay (Pierce, Rockford, IL) to guide equal loading of each sample to 6% to 15% SDS-polyacrylamide gels. Resolved proteins are transferred to PVDF membranes, probed with proper primary and secondary antibodies, and visualized by the Western Lighting Plus ECL kit (Perkin Elmer, Waltham, MA). The film images are scanned and densitometry determined with the Quantity One Software (Bio-Rad). The antibodies against murine and human glycolytic enzymes are purchased from Cell Signaling Technology.
For measuring the activities of individual glycolytic enzymes, whole cell lysates are prepared and the activity of each glycolytic enzyme assayed as above-described.
For immuno-blot assays, we employ a reported method53 (link). Cells are lysed in 625 mM Tris-HCl (pH 6.8), 10% SDS, 25% glycerol, 5% β-mercaptoethanol and 0.015% bromphenol blue supplemented with a protease inhibitor cocktail (Pierce, Rockford, IL), followed by sonication. Samples are stored at −80°C after heating and protein concentrations scored by the BCA protein assay (Pierce, Rockford, IL) to guide equal loading of each sample to 6% to 15% SDS-polyacrylamide gels. Resolved proteins are transferred to PVDF membranes, probed with proper primary and secondary antibodies, and visualized by the Western Lighting Plus ECL kit (Perkin Elmer, Waltham, MA). The film images are scanned and densitometry determined with the Quantity One Software (Bio-Rad). The antibodies against murine and human glycolytic enzymes are purchased from Cell Signaling Technology.
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