A549 cells were harvested 24–60 h after cytokine stimulation depending on the parameters of the experiment. After washing with phosphate-buffered saline (PBS), cells were directly lysed in 2 × sodium dodecyl sulfate(SDS) sample buffer without bromophenol blue dye (100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 2% β-mercaptoethanol, 25 mM ethylenediaminetetraacetic acid (EDTA)). Protein concentration was determined using a protein quantification assay kit (Macherey-Nagel, Duren, Germany). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes. After blocking with 2% ECL advance blocking reagent™ (GE Healthcare, NJ, USA) in PBS containing 0.1% Tween 20 (polyoxyethelenesorbitan monolaurate), the membranes were incubated overnight with primary antibodies at 4 °C. After washing and incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG, the antigen-antibody complexes were detected using chemiluminescence (ECL select detection system™, GE Healthcare) and then visualized using an Imagequant Las4000 system™ (Bio-Rad, CA, USA).
Ecl advance blocking reagent
Manufactured by GE Healthcare
Sourced in United States
The ECL Advance blocking reagent is a laboratory product designed to block non-specific binding sites in Western blotting and related immunoassay techniques. Its core function is to prevent background signals and improve the specificity of target protein detection.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Cytiva (3 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (2 mentions)
Protein quantification assay kit, by Macherey-Nagel (1 mentions)
Ecl select detection system, by GE Healthcare (1 mentions)
Phosphatase inhibitor cocktail, by Selleck Chemicals (1 mentions)
Protease inhibitor cocktail, by Selleck Chemicals (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Anti neun, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Transfer apparatus, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura, by Thermo Fisher Scientific (1 mentions)
0.1 mm zirconium beads, by Biospec (1 mentions)
Tissuelyser, by Qiagen (1 mentions)
Versadoc imaging system, by Bio-Rad (1 mentions)
Hybond ecl transfer membrane, by GE Healthcare (1 mentions)
Ecl detection reagent, by GE Healthcare (1 mentions)
Nbt bcip solution, by Roche (1 mentions)
Dig labeling kit, by Roche (1 mentions)
Costar 96 well eia ria plates, by Corning (1 mentions)
10 protocols using ecl advance blocking reagent
1
Western Blot Analysis of Cytokine-Stimulated Cells
2
Protein Extraction and Immunoblotting Protocol
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Cells were lysed by incubation in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris–HCl (pH 8.0) supplemented with Protease Inhibitor Cocktail (Bimake) and Phosphatase Inhibitor Cocktail (Bimake) for 30 min on ice. Next, the cell extracts were sonicated with a VirSonic 100 ultrasonic cell disrupter and stored at −70°C. The protein concentration was measured by the Bradford assay. Aliquots of each sample were separated by sodium dodecyl sulphate-polyacrylamide gelelectrophoresis and blotted onto polyvinylidene difluoride (PVDF) membranes (Amersham/GE Healthcare). The membranes were blocked for 1 h in 2% ECL Advance blocking reagent (GE Healthcare) or 2% bovine serum albumin (BSA) (Sigma) in PBS containing 0.1% Tween 20 (PBS-T) followed by incubation overnight at 4°C with a primary antibody diluted in PBS-T containing 2% blocking reagent or 2% BSA. After three washes with PBS-T, the membranes were incubated for 1 h with the secondary antibodies (horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG) in PBS-T containing 2% blocking agent or 2% BSA. The immunoblots were visualized using a Pierce ECL plus western blotting substrate.
3
Immunoblot Analysis of Brain Proteins
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Immunoblot analysis using brain tissues was performed as described previously (Arimura et al., 2012 (link)). Membranes were incubated for 1 h with ECL Advance blocking reagent (GE Healthcare #RPN418) at room temperature, and probed overnight at 4°C with primary antibodies: anti-PDGFRβ (1:1000; CST #3169), anti-β-actin (1:3000; Sigma-Aldrich #A5441), anti-p-AKT (1:1000; CST #4060), anti-pan-AKT (1:1000; CST #4691), anti-p-ERK (1:1000; CST #4370), anti-ERK (1:1000; CST #4695), anti-p-STAT3 (1:1000; CST #9145), anti-STAT3 (1:1000; CST #4904), anti-GFAP (1:1000; CST #3670), and anti-NeuN (1:1000; Millipore #MAB377). Membranes were then washed and incubated with secondary antibodies (1:100,000; CST #7074 or #7076) for 45 min at room temperature.
4
HeLa Cell Lysis and Western Blot
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HeLa cells were lysed by incubation in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol and 1 mM PMSF) supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich) and Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich) for 30 min on ice. Next, the cell extracts were sonicated with a VirSonic 100 ultrasonic cell disrupter and stored at −70°C. The protein concentration was measured by the Bradford assay. Aliquots of each sample were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride (PVDF) membranes (Amersham/GE Healthcare). The membranes were blocked for 1 h in 2% ECL Advance blocking reagent (GE Healthcare) in PBS containing 0.1% Tween 20 (PBS-T) followed by incubation overnight with a primary antibody diluted in PBS-T containing 2% blocking reagent or 1% BSA. After three washes with PBS-T, the membranes were incubated for 1 h with the secondary antibodies (horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG) in PBS-T containing 2% blocking agent or 1% BSA. The immunoblots were visualised using a Pierce ECL plus western blotting substrate. The films were scanned and processed with Adobe Photoshop CS6 software.
5
Immunoblotting of Synechocystis Proteins
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Synechocystis cells were resuspended in 100 μl of 10 mm Tris-HCl, pH 7.3, and heated for 5 min at 100 °C followed by centrifugation at 10,000 rpm for 5 min at 4 °C. The samples were analyzed by SDS-PAGE on 4–12% NuPAGE acrylamide gradient gels (Life Technologies). Protein was blotted onto nitrocellulose membrane, which was blocked for 1 h in Tris-buffered saline with Tween 20 (TSB-T) and 2% ECL Advance blocking reagent (GE Healthcare). Antibody incubations (1 h) and membrane washes were performed in TBS-T. Anti-IdiA and anti-Slr0513 (FutA2) antisera were a kind gift from E. Pistorius (24 (link), 34 (link)) and were used at concentrations of 1:5000 and 1:10000 respectively. Following application of ECL Advance detection reagent (GE Healthcare), chemiluminescence was visualized using a VersaDocTM CCD imager (Bio-Rad).
6
Whole-Cell Extract Preparation and Western Blot Analysis
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S. elongatus whole-cell extracts were prepared as described previously (22 (link)), with minor modifications. Cells were disrupted using a tissue lyser (Qiagen) with zirconium beads (0.1 mm; Biospec Products) for two cycles of 30 s at a frequency of 30 Hz. After centrifugation, the supernatants were used to determine protein concentration by the BCA method using bovine serum albumin (BSA) as a standard. Next, 30 µg of total protein was separated in a Bis-Tris NuPAGE gel and transferred to a 0.45-μm polyvinyl fluoride (PVDF) membrane using a transfer apparatus according to the manufacturer’s protocols (Invitrogen). After incubation with blocking solution (TBS-T; 20 mM Tris-Cl, 150 mM NaCl, 0.02% [vol/vol] Tween-20, pH 7.6 supplemented with 2% [wt/vol] ECL Advance blocking reagent; GE Healthcare) for 1 h, the membrane was washed once, and incubated with mouse monoclonal anti-FLAG M2-peroxidase (HRP) antibody (Sigma; 1:1000). Membranes were washed three times for 5 min in TBS-T then developed with the SuperSignal West Dura (Thermo Scientific) ECL system and imaged using a Versa-Doc Imaging system (Bio-Rad).
7
Western Blot Protein Detection Protocol
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Cells were collected and either snap frozen in liquid nitrogen or immediately lysed using 2 × Laemmli buffer. Protein amounts were normalized using known concentrations of BSA and protein absorbance was measured using Cary 60 Spectrophotometer technology. SDS-gels were run at 15–18 mA and proteins were either wet-blotted overnight (30 V, 4 °C) or for 2 h (100 V, 4 °C) on Hybond ECL transfer membrane (GE Healthcare). Membranes were blocked in 2% ECL Advance Blocking Reagent (GE Healthcare) in 0.1% TBST (1 × TBS supplemented with 0.1% Tween 20) for at least 30 min and incubated with primary antibodies overnight at 4 °C or at room temperature for 4 h in blocking solution and secondary antibodies were added for 1 h at room temperature (in blocking solution). Membranes were washed three times with 0.1% TBST, 10' each, after primary and secondary antibody incubations and detected with ECL detection reagent (GE healthcare). Differences in protein levels were normalized against the loading control and the signal intensity was quantified using ImageJ. For complete, uncropped blots with size markers, see Supplementary Fig. 8 .
8
Localization of NADPH Oxidase mRNAs in Seeds
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To examine the localization of NADPH oxidase mRNAs in seeds, we performed tissue printing according to the method of Nonogaki et al. [40 (link)]. After being soaked for 24 h in water, seeds were longitudinally sliced in two with a razor blade. The cut surfaces were pressed onto a Hybond-N+ membrane for 15 s. The membrane was cross-linked under UV light and hybridized with RNA probes (both sense and antisense). The RNA probes were prepared from PCR products by using NADPH oxidase common primers [9 ] in a digoxigenin (DIG) labeling kit (Roche Diagnostics). The membrane was prehybridized at 65°C for 1 h in 0.3 M phosphate buffer containing 7% SDS, and then hybridized by incubation in the same buffer with DIG-labeled probes at 65°C for over 15 h. The membrane was then washed in 2× SSC containing 0.1% SDS (15 min), and then in 0.1× SSC containing 0.1% SDS (15 min) at 70°C. It was then blocked with ECL Advance blocking reagent (GE Healthcare) for 1 h and incubated with alkaline phosphatase—conjugated anti-DIG antibody for 1 h at 37°C. Signals were colorimetrically detected by using NBT/BCIP solution (Roche Diagnostics).
9
Western Blot Analysis of Protein Extracts
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HeLa cells were lysed by incubation in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol, and 1 mM PMSF) supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich) and Phosphatase Inhibitor Cocktail 2 (Sigma-Aldrich) for 30 min on ice. Next, the cell extracts were sonicated with a VirSonic 100 ultrasonic cell disrupter and stored at −70°C. The protein concentration was measured by the Bradford assay. Aliquots of each sample were separated by SDS–PAGE and blotted onto PVDF membranes (Amersham/GE Healthcare). The membranes were blocked for 1 hr in 2% ECL Advance blocking reagent (GE Healthcare) in PBS containing 0.1% Tween 20 (PBS-T) followed by overnight incubation with a primary antibody diluted in PBS-T containing 2% blocking reagent. After three washes with PBS-T, the membranes were incubated for 1 hr with the secondary antibodies (horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG) in PBS-T containing 2% blocking agent. The immunoblots were visualised using a Pierce ECL plus western blotting substrate.
10
ELISA for Antibody Titer Determination
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The overnight coating of Costar 96 Well EIA/RIA plates (Corning) was carried out at 4 °C with 100 µL of 4 µg/mL RBD (RayBiotech, or BSA (used as control) in Coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.8). After 4 washes with 300 µL of PBS, the plate was blocked with 200 µl of a 2% (w/v) ECL Advance Blocking Reagent (GE Healthcare) solution in PBS-T (PBS supplemented with 0.1% (v/v) Tween-20) for 2 h at RT. The plate was washed 4 times with PBS, and then, starting at 2 µg of the purified antibody per well (100 µl), 1:5 serial dilutions in blocking solution were incubated for 1 h 30 min at RT. After 4 washing steps with PBS-T (PBS buffer supplemented with 0.05% Tween-20), 1:2000 HRP-labelled rabbit anti-human IgG (Sigma-Aldrich, #A8792) in blocking solution was added. After 1 h, the plate was washed with PBS and the substrate o-phenylenediamine dihydrochloride SIGMAFAST™ OPD tablet (Sigma-Aldrich, #P9187) was added (following manufacturer's instructions). Reactions were stopped with 50 µL 3 M HCl per well and absorbance was measured at 492 nm. The endpoint titer was determined as the last concentration of each purified antibody showing an absorbance value higher than the value defined as cutoff (mean blank + 3SD). Blank is defined as the values from each ELISA test against BSA (Zrein et al., 1986; Armbruster and Pry, 2008) .
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