Cells were washed with cold PBS and lysed in 2X Laemmli Sample Buffer (Bio-Rad Laboratories, 1,610,737). Whole-cell lysates were subjected to SDS-PAGE on 4–20% gradient gels (mini-PROTEAN TGX; Bio-Rad). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (TransBlot® TurboTM LF PVDF, Bio-Rad) followed by blocking in 3% BSA and antibody incubation in Tris-buffered saline with 0.1% Tween-20 (Sigma-Aldrich, P1379). Membranes incubated with fluorescent secondary antibodies (IRDye 680 rabbit, 926–68,073; IRDye 680 mouse, 926–68,072; IRDye 800 rabbit, 926–32,213; IRDye 800 mouse, 926–32,212)) were developed with an Odyssey infrared scanner (LI-COR Biosciences), whereas those incubated with HRP (horseradish peroxidase)–conjugated antibodies (HRP rabbit, 111 035 144; HRP mouse, 115 035 146) were developed using Clarity western ECL substrate solutions (Bio-Rad) with a ChemiDoc XRS+ imaging system (Bio-Rad).
Clarity western ecl substrate solution
Manufactured by Bio-Rad
Sourced in Canada
Clarity Western ECL substrate solutions are chemiluminescent detection reagents used for the visualization of proteins in Western blot analysis. The solutions contain the necessary components to generate a luminescent signal when interacting with the reporter enzyme conjugated to the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xrs imaging system, by Bio-Rad (9 mentions)
Odyssey infrared scanner, by LI COR (6 mentions)
Transblot turbo lf pvdf, by Bio-Rad (5 mentions)
Mini protean tgx, by Bio-Rad (5 mentions)
Irdye 800, by LI COR (5 mentions)
Irdye 680, by LI COR (5 mentions)
Immobilon p membrane, by Merck Group (4 mentions)
Laemmli sample buffer, by Bio-Rad (3 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Trans blot turbo transfer system, by Bio-Rad (2 mentions)
Potassium acetate, by Merck Group (1 mentions)
Magnesium acetate, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Hepes, by Merck Group (1 mentions)
Np 40, by Merck Group (1 mentions)
H4034, by Merck Group (1 mentions)
Donkey polyclonal anti rabbit igg hrp, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Santa Cruz Biotechnology (1 mentions)
17 protocols using clarity western ecl substrate solution
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Protein Lysates
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Cells were lysed in 2× sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 200 mM DTT, and 0.004% bromophenol blue). Whole-cell lysates were subjected to SDS-PAGE on 10% or 4–20% gradient gels (mini-PROTEAN TGX; Bio-Rad). Proteins were transferred to Immobilon-P membranes (Millipore) followed by blocking and antibody incubation in 5% fat-free milk powder in Tris-buffered saline with 0.05% Tween 20. Membranes incubated with fluorescent secondary antibodies (IRDye680 and IRDye800; LI-COR) were developed with an Odyssey infrared scanner (LI-COR), whereas those incubated with horseradish peroxidase–conjugated antibodies were developed using Clarity Western ECL substrate solutions (Bio-Rad) with a ChemiDoc XRS+ imaging system (Bio-Rad).
3
Western Blot Protocol with Quantification
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Cells were washed with ice-cold PBS and lysed with 25 mM Hepes, pH 7.2 (H4034; Sigma-Aldrich), 125 mM potassium acetate (104820; Merck Millipore), 2.5 mM magnesium acetate (105819; Merck Millipore), 5 mM EGTA (E3889; Sigma-Aldrich), 0.5% Triton-X-100 (Sigma-Aldrich) and 1 mM dithiothreitol (DTT; D0632; Sigma-Aldrich) supplemented with protease inhibitor cocktail (P9340; Sigma-Aldrich) or lysed in 2× sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 200 mM DTT and 0.004% bromophenol blue). Lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10% or 4–20% gradient gels (mini-PROTEAN TGX; Bio-Rad). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (TransBlot® TurboTM LF PVDF, Bio-Rad) followed by antibody incubation in 2% bovine serum albumin in Tris-buffered saline with 0.1% Tween-20. Membranes incubated with fluorescent secondary antibodies (IRDye680 or IRDye800; LI-COR) were developed with an Odyssey infrared scanner (LI-COR), whereas those incubated with horseradish peroxidase-conjugated antibodies were developed using Clarity Western ECL substrate solutions (Bio-Rad) with a ChemiDoc XRS+ imaging system (Bio-Rad). Quantification of immunoblots was done using the Odyssey Software. Please see Supplementary Figs. 9 –11 for uncropped membranes.
4
Western Blot Protein Analysis
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Cells were washed with ice-cold PBS and lysed with 25 mM Hepes, pH 7.2 (H4034; Sigma-Aldrich), 125 mM potassium acetate (104820; Merck Millipore), 2.5 mM magnesium acetate (105819; Merck Millipore), 5 mM EGTA (E3889; Sigma-Aldrich), 0.5% NP-40 (I18896; Sigma-Aldrich), and 1 mM DTT (D0632; Sigma-Aldrich) supplemented with protease inhibitor cocktail (P9340; Sigma-Aldrich). Cell lysates were then subjected to SDS-PAGE on 10% (567-1034; Bio-Rad) or 4%–20% (567-1094; Bio-Rad) gradient gels and blotted onto Immobilon-P membranes (IPVH00010; Merck Millipore). Membranes incubated with fluorescently labeled secondary antibodies (IRDye680 and IRDye800; LI-COR) were developed by Odyssey infrared scanner (LI-COR). Membranes detected with HRP-labeled secondary antibodies were developed using Clarity Western ECL substrate solutions (Bio-Rad) with a ChemiDoc XRS+ imaging system (Bio-Rad). Quantification of immunoblots was performed using the Odyssey, Volume tools of Image Lab (Bio-Rad), or ImageJ software (National Institutes of Health).
5
Western Blot Imaging and Quantification
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Samples were subjected to SDS-PAGE on 10% (567–1034; Bio-Rad) or 4–20% (567–1094; Bio-Rad) gradient gels and blotted onto Immobilon-P membranes (IPVH00010; Merck Millipore). Membranes incubated with fluorescently labelled secondary antibodies (IRDye680 and IRDye800; LI-COR) were developed by Odyssey 3.0.30 infrared scanner (LI-COR) or Azure Sapphire Biomolecular Imager. Membranes detected with HRP-labelled secondary antibodies were developed using Clarity Western ECL substrate solutions (Bio-Rad) with a ChemiDoc XRS+ imaging system (Bio-Rad) or Azure Sapphire Biomolecular Imager. Quantification of WB data was performed with ImageJ/Fiji 1.52p, Odyssey 3.0.30 or Azure Spot 2.1.097 (Azure Biosystems) and samples were normalized to a loading control (Actin, GAPDH or Vinculin). Uncropped Western blot images are displayed in Supplementary Fig. 13 .
6
Western Blot Analysis of Cell Lysates
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Cells were washed with cold PBS and lysed in 2× sample buffer (125 mM Tris–HCl, pH 6.8, 4% SDS, 20% glycerol, 200 mM DTT, and 0.004% bromophenol blue). Whole‐cell lysates were subjected to SDS–PAGE on 4–20% gradient gels (Mini‐PROTEAN TGX; Bio‐Rad). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Trans‐Blot® Turbo™ LF PVDF, Bio‐Rad) followed by blocking in 5% fat‐free milk powder and antibody incubation in 5% fat‐free milk powder in Tris‐buffered saline with 0.1% Tween‐20. Membranes incubated with horseradish peroxidase‐conjugated antibodies were developed using Clarity Western ECL Substrate Solutions (Bio‐Rad) with a ChemiDoc XRS+ imaging system (Bio‐Rad).
7
Western Blot Protein Analysis
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Protein samples were subjected to gel electrophoresis on 10% or 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Proteins were transferred to nitrocellulose membrane membranes (Bio-Rad) followed by blocking in PBS-T (0.05% Tween-20) with 5% milk powder and antibody incubation. Membranes incubated with horseradish peroxidase-conjugated antibodies were developed using Clarity Western ECL substrate solutions (Bio-Rad). Quantification of immunoblots was done using the ImageJ Software. The uncropped western blot images used in this paper can be seen in Supplementary Figures 11 –17 .
8
SDS-PAGE and Western Blot Analyses
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Cells were washed with ice-cold PBS and lysed in 2× sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 200 mM DTT, and 0.004% bromophenol blue). Cell lysates were then subjected to SDS-PAGE on 10% (567–1034; Bio-Rad) or 4–20% (567–1094; Bio-Rad) gradient gels and blotted onto Immobilon-P membranes (IPVH00010; Merck Millipore). Membranes incubated with fluorescently labeled secondary antibodies (IRDye680 and IRDye800; LI-COR) were developed by Odyssey infrared scanner (LI-COR). Membranes detected with HRP-labeled secondary antibodies were developed using Clarity Western ECL substrate solutions (Bio-Rad) with a ChemiDoc XRS+ imaging system (Bio-Rad).
9
Western Blot Protein Analysis Protocol
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Western blot was performed using standard methods. In brief, cells were washed with PBS and lysed with sample buffer (100 mM Tris-HCl 6.8, 4% SDS, 20% glycerol, 0.2 M DTT) followed by boiling 5 min at 95°C and vortexing to shear genomic DNA. After SDS-PAGE, proteins were transferred onto a nitrocellulose membrane (Santa Cruz Biotechnology) by tank transfer. Primary antibodies were incubated overnight at 4°C, secondary antibodies for an hour at room temperature. All western blots were developed with Clarity Western ECL substrate solutions (Bio-Rad). Antibodies were as follows: Rabbit polyclonal anti-GFP (in house; 1:2000), Mouse monoclonal anti-HA (HRP) (Roche; 1:5000), Donkey polyclonal anti-rabbit IgG (HRP) (GE Healthcare).
10
Western Blot Analysis of Protein Expression
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Cells were washed in cold PBS and lysed in 2× Laemmli sample buffer (Bio-Rad Laboratories, 1610737) supplemented with dithiothreitol (DTT). Whole-cell lysates were separated by SDS–PAGE on 4–20% gradient gels (mini-PROTEAN TGX; Bio-Rad). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes (TransBlot TurboTM LF PVDF, Bio-Rad) followed by 1 h blocking in 3% BSA and overnight antibody incubation in Tris-buffered saline with 0.1% Tween-20 (Sigma-Aldrich, P1379) at 4°C. Membranes incubated with HRP (horseradish peroxidase)-conjugated antibodies (HRP-conjugated anti-rabbit IgG, 111 035 144; HRP-conjugated anti-mouse IgG, 115 035 146; both Jackson ImmunoResearch) were developed using Clarity western ECL substrate solution (Bio-Rad) with a ChemiDoc XRS+ imaging system (Bio-Rad).
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