Total protein was extracted from the cell pellets using the NucleoSpin RNA/Protein Kit (Macherey‐Nagel, Dueren, Germany), and concentration determined using the Protein Quantification Assay Kit (Macherey‐Nagel) and a microplate photometer (Multiskan EX, Thermo Scientific, Waltham, MA, USA). Equal amounts of protein (10 μg) were resolved by SDS‐PAGE. Band intensity, mirroring protein levels were visualized using chemiluminescence detection systems Supersignal West Pico (Thermo Scientific) or Substrat HRP Immobilon Western (Merck Millipore) following the manufacturers’ instructions.
Substrat hrp immobilon western
Manufactured by Merck Group
Sourced in United States
Substrat HRP Immobilon Western is a lab equipment product. It is a substrate used in Western blotting techniques, which detect and quantify specific proteins in a sample. The product facilitates the chemiluminescent detection of horseradish peroxidase (HRP) conjugated to secondary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Tgx gel, by Bio-Rad (4 mentions)
Transturbo system, by Bio-Rad (4 mentions)
Anti flag m2 monoclonal antibody peroxidase conjugate, by Merck Group (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna protein kit, by Macherey-Nagel (1 mentions)
Multiskan ex, by Thermo Fisher Scientific (1 mentions)
Protein quantification assay kit, by Macherey-Nagel (1 mentions)
Flag m2 magnetic beads, by Merck Group (1 mentions)
Mini protean tgx stain free gel, by Bio-Rad (1 mentions)
Mouse anti his antibody, by Cell Signaling Technology (1 mentions)
Chemidoc imager, by Bio-Rad (1 mentions)
Mouse anti ha antibody, by Cell Signaling Technology (1 mentions)
Chemigenius2, by Syngene (1 mentions)
Peroxidase coupled goat anti rabbit secondary antibody, by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
13 protocols using substrat hrp immobilon western
1
Protein Quantification and Visualization
2
Immunoprecipitation of FLAG-tagged Proteins
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0.5 g of 3-week-old seedling tissue were ground in liquid nitrogen, resuspended in 1.5mL ice-cold IP buffer [50mM Tris pH 7.6, 150mM NaCl, 5mM MgCl2, 0.1% Nonidet P-40, 10% glycerol, 0.5 mM DTT, 1x Protease Inhibitor Mixture (Roche)], and centrifuged 2 times for 15 min at 4°C, 16 000g. 50μL M2 magnetic FLAG-beads (Sigma, M8823) were added to the supernatants and incubated for 2 hour rotating at 4°C. Beads were washed 3 times in ice-cold IP buffer for 10 min rotating at 4°C. Immunoprecipitated proteins were denatured in Laemmli buffer for 5min at 95°C. 10μL of input and bead elution were run on 10% SDS-PAGE gels, and proteins were detected by western blotting using either Anti-FLAG M2 monoclonal antibody-peroxidase conjugate (Sigma, A8592) at a dilution of 1:10000, or c-Myc rat monoclonal antibody (Chromotek, 9E1-100) at a dilution of 1:1000 followed by goat anti-rat IgG horseradish peroxidase (Abcam, ab205720) used at a dilution of 1:20000 as secondary antibody. Western blots were developed using Substrat HRP Immobilon Western (Merck Millipore, WBKLS0500).
3
Western Blot Analysis of HA and His-Tagged Proteins
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SDS-PAGE was performed under reducing conditions on Mini-PROTEAN TGX Stain-Free gels (BioRad, USA). Proteins were then transferred onto a Trans-Blot Turbo PVDF Western blotting membrane (BioRad, USA). Antibody dilutions were 1:1,000 for the mouse anti-HA antibody (catalogue number: 2367, Cell Signaling, USA), mouse anti-His antibody (catalogue number: 2366, Cell Signaling, USA) and secondary anti-mouse horseradish peroxidase (HRP)-linked antibody (catalogue number: 7076, Cell Signaling, USA). Signals were visualized using the Substrat HRP Immobilon Western (Merck Millipore, USA) and a ChemiDoc imager (BioRad, USA).
4
Protein Extraction and Western Blot
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Total proteins were extracted from leaves of 3-wk-old seedlings using 8 M urea and denatured in Laemmli buffer for 5 min at 95°C. 10–15 μl of protein extracts were run on 10% SDS–PAGE, and proteins were detected by Western blotting using Anti-FLAG M2 monoclonal antibody–peroxidase conjugate (A8592; Sigma-Aldrich) at a dilution of 1:10,000. Western blots were developed using Substrat HRP Immobilon Western (WBKLS0500; Merck Millipore).
5
Western Blot Imaging Protocol
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Proteins separated on a 12.5 % SDS-PAGE were transferred to a nitrocellulose membrane.
The membrane was blocked with 2% skim milk in 0.1% TBS-Tween (TBS-T) and incubated overnight at 4°C with the indicated antibody. After three 10min washes in TBS-T, the membrane was incubated with the peroxidase-coupled goat antirabbit secondary antibody (1/2000; Sigma) for 2 hours in TBS-T-2% skim milk at RT. After three more washes with TBS-T, the membrane was revealed using a chemiluminescent substrate (Substrat HRP Immobilon Western, Merck-Millipore) and imaged with a digital analyzer (ChemiGenius2; Syngene).
The membrane was blocked with 2% skim milk in 0.1% TBS-Tween (TBS-T) and incubated overnight at 4°C with the indicated antibody. After three 10min washes in TBS-T, the membrane was incubated with the peroxidase-coupled goat antirabbit secondary antibody (1/2000; Sigma) for 2 hours in TBS-T-2% skim milk at RT. After three more washes with TBS-T, the membrane was revealed using a chemiluminescent substrate (Substrat HRP Immobilon Western, Merck-Millipore) and imaged with a digital analyzer (ChemiGenius2; Syngene).
6
Dystrophin Protein Expression Analysis
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To confirm the presence of dystrophin protein expression, Western blots were performed. Total protein was extracted from TA muscle samples with lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 % Triton, 1 % sodium deoxycholate, 0.1 % SDS, and Complete Protease inhibitor cocktail (Roche)) and quantified using BCA Protein Assay Kit (Thermo Scientific Pierce). After a denaturation step for 5 min at 95 °C, 50 μg of total protein extract was loaded in Novex 4–12 % Bis-Tris protein gels (Life Technologies) and transferred to nitrocellulose membrane. Blots were blocked for 1 h with 10 % non-fat milk in Tris-buffered saline. Dystrophin and alpha-actinin proteins were detected by probing the membrane with 1:100 dilution of monoclonal NCL-DYS-1 primary antibody (Novocastra) and 1:1000 of monoclonal anti-alpha-actinin primary antibody (Sigma), respectively. An incubation with 1:5000 of sheep anti-mouse secondary antibody (horseradish peroxidase conjugated) allowed visualisation using Substrat HRP Immobilon Western (Millipore). Band intensities were analyzed using ImageJ software.
7
Protein Analysis by Immunoblotting
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Protein analysis by immunoblotting was performed essentially as previously described27 (link). Briefly, samples were collected, washed out with PBS and lysed with RIPA buffer. Protein concentration was determined by BCA assay (Pierce) before electrophoresis in 4–15% TGX gels (BioRad) and equal amount of protein was loaded in each well. Protein transfer was performed in TransTurbo system (BioRad) in PVDF membranes. After blocking for 1 h with 5% non-fat milk, membranes were incubated overnight at 4 °C in agitation with primary antibodies, washed three times with PBS-Tween 0,1% and incubated with the appropriate HRP-labeled secondary antibody for 1 h. Membranes were washed out three times with PBS-Tween 0,1% and developed with Substrat HRP Immobilon Western (Millipore). Band quantification was performed using the “ImageLab” software from BioRad and represented as the ratio between the protein of interest and a control protein i.e. actin. The value of 1 is arbitrarily given to control cells. One blot representative of several experiments is shown.
8
Immunoblotting for Protein Analysis
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Protein analysis by immunoblotting was performed essentially. Briefly, samples were collected, washed out with PBS and lysed with RIPA buffer. Protein concentration was determined by BCA assay (Pierce; Dallas, TX, USA) before electrophoresis in 4–15% TGX gels (BioRad) and equal amount of protein was loaded in each well. Protein transfer was performed in TransTurbo system (BioRad) in PVDF membranes. After blocking for 1 h with 5% non-fat milk, membranes were incubated overnight at 4 °C in agitation with primary antibodies, washed three times with PBS-Tween 0.1% and incubated with the appropriate HRP-labeled secondary antibody for 1 h. Membranes were washed out three times with PBS-Tween 0.1% and developed with Substrat HRP Immobilon Western (Millipore). Band quantification was performed using the “ImageLab” (version 3.0.1) software from Bio-Rad and represented as the ratio between the protein of interest and a control protein, i.e., actin. The value of 1 is arbitrarily given to control cells. One blot representative of several experiments is shown.
9
Immunoblotting Protein Analysis Protocol
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Protein analysis by immunoblotting was performed essentially as previously described (Garaude et al., 2006 (link), Garaude et al., 2008 (link)). Briefly, samples were collected, washed out with PBS and lysed with RIPA buffer. Protein concentration was determined by BCA assay (Pierce) before electrophoresis in 4–15% TGX gels (BioRad) and equal amount of protein was loaded in each well. Protein transfer was performed in TransTurbo system (BioRad) in PVDF membranes. After blocking for 1 h with 5% non-fat milk, membranes were incubated overnight at 4 °C in agitation with primary antibodies, washed three times with TBS-Tween 0,1% and incubated with the appropriate HRP-labeled secondary antibody for 1 h. Membranes were washed out three times with TBS-Tween 0,1% and developed with Substrat HRP Immobilon Western (Millipore). Band quantification was performed using the “ImageLab” software from BioRad and represented as the ratio between the protein of interest and a control protein i.e. actin. The value of 1 is arbitrarily given to control cells. One blot representative of several experiments is shown.
10
Immunoblotting for Protein Analysis
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Protein analysis by immunoblotting was performed essentially as previously described45 (link). Briefly, samples were collected, washed out with PBS and lysed with RIPA buffer. Protein concentration was determined by BCA assay (Pierce) before electrophoresis in 4–15% TGX gels (BioRad) and equal amount of protein was loaded in each well. Protein transfer was performed in TransTurbo system (BioRad) in PVDF membranes. After blocking for 1 h with 5% non-fat milk, membranes were incubated overnight at 4 °C in agitation with primary antibodies, washed three times with PBS-Tween 0,1% and incubated with the appropriate HRP-labeled secondary antibody for 1 h. Membranes were washed out three times with PBS-Tween 0,1% and developed with Substrat HRP Immobilon Western (Millipore). Band quantification was performed using the “ImageLab” software from BioRad and represented as the ratio between the protein of interest and a control protein i.e. actin. The value of 1 is arbitrarily given to control cells. One blot representative of several experiments is shown.
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