Western blot analysis of total cell lysates was performed as previously described [49 (link)]. In detail, total cellular proteins were obtained by lysing the cells with lysis buffer (50 mM Tris-Cl, pH 7.5; 150 mM NaCl; 1% w/v Nonidet p-40; 0.5% w/v sodium deoxycholate, 0.1% w/v SDS, 1 mM EDTA) (EDTA, Ethylenediaminetetraacetic acid) in the presence of proteases and phosphatase inhibitors. Equal amounts of protein were separated by SDS polyacrylamide gel electrophoresis, blotted to PVDF (Polyvinylidene fluoride), and transferred using the Trans-Blot Turbo Transfer System (Bio-Rad) with 25 V, 1.0 A, for 30 min. After blocking in 5% non-fat milk (Bio-Rad), membranes were probed with the following primary antibodies: anti-PTEN (1:1000, clone 6H2.1; Agilent Technologies, Santa Clara, CA, USA), anti-p-mTOR (1:1000; Cell Signaling Technology, Danvers, MA), anti-mTOR (1:1000, clone 7C10; Cell Signaling Technology), and anti- β-actin (1:5000, clone AC-15; Sigma-Aldrich), at 4 °C, overnight. After incubation with secondary horseradish peroxidase-conjugated antibodies (Bio-Rad), specific proteins were visualized by the enhanced chemiluminescence system (Amersham Biosciences, Buckinghamshire, UK) using a ChemiDoc XRS+ imaging system (Bio-Rad).
Secondary horseradish peroxidase conjugated antibodies
Manufactured by Bio-Rad
Sourced in United Kingdom, United States
Secondary horseradish peroxidase-conjugated antibodies are antibodies that have been conjugated with the enzyme horseradish peroxidase. This enzyme can be used as a label to detect the presence of a target molecule in various assays, such as Western blotting, ELISA, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
Non fat milk, by Bio-Rad (1 mentions)
Enhanced chemiluminescence system, by Cytiva (1 mentions)
Anti mtor, by Cell Signaling Technology (1 mentions)
Anti p mtor, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
Immobilon western, by Merck Group (1 mentions)
Streptavidin hrp, by Agilent Technologies (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
5 protocols using secondary horseradish peroxidase conjugated antibodies
1
Western Blot Analysis of Total Cell Lysates
2
GST-Fusion Protein Pulldown Assay
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Equal amounts of recombinant GST and GST–desmin, GST–NDUFS2 or GST–prosaposin were bound to glutathione Sepharose and mixed with either 0.1 mg of desmin-enriched cardiac extract or 1 mg of total protein cardiac extracts or total extracts from transfected COS-7 cells, at room temperature for 2 h. Bound proteins, after washing in the cold washing buffer (10 mM Tris, pH 8.5, 0.01% (v/v) Nonidet P-40, 0.01% (v/v) DOC, 5 mM EDTA and 2 mM DTT) were eluted by heating for 5 min at 90 °C in SDS-PAGE sample buffer (50 mM Tris, pH 6.8, 50 mM DTT, 2% (v/v) SDS, 0.2% (v/v) bromophenol blue, 10% (v/v) glycerol). The soluble fractions were analyzed by SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with desmin polyclonal antibody Y-20, (Santa Cruz Biotechnology, Heidelberg, Germany) or anti-NDUFS2 (#PA5-19342, from Thermo Fisher Scientific, Waltham, MA, USA) at a 1:300 dilution. Secondary horseradish peroxidase-conjugated antibodies were from BioRad (Hercules, CA, USA) and visualization of the peroxidase was performed with enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ, USA).
3
Western Blot Analysis of Piezo1, Rac1, and GAPDH
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Cells were rinsed in PBS, suspended in lysis buffer (50 mM Tris-HCl buffer, pH 8.0, containing 150 mM NaCl, 1% Triton X-100, 0.05% sodium deoxycholate, 10 mM EDTA, and protease and phosphatase inhibitors), and cleared by centrifugation. The lysates were then reduced in Laemmli sample buffer at 95°C, separated by polyacrylamide gel electrophoresis in the presence of SDS, and transferred onto PVDF membranes. Membranes were blocked with 5% milk powder in Tris-buffered saline containing 0.05% Tween (TBS-Tween) for 1 h at room temperature, after which primary antibodies were added in 5% BSA, TBS-Tween and incubated overnight at +4°C. The membranes were subsequently washed in TBS-Tween, after which secondary horseradish peroxidase conjugated antibodies (Bio-Rad) were added in 5% milk powder in TBS-Tween and incubated for 30 min at room temperature. After extensive washing in TBS-Tween, antibody binding was detected by chemiluminescence (Immobilon Western, Millipore) using the Bio-Rad ChemiDoc Imaging System. The following antibodies were used: Piezo1 (Novus Biol. NBP1-78537;1:1000), GAPDH (Cell Signaling; 2118L;1:5000), Rac1 (Cytoskeleton ARC03; 1:1000), Streptavidin-HRP (DAKO P0397; 1:1000).
4
Western Blot Analysis of Cell Lysates
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Western blot analysis of total cell lysates was performed as previously described [6 (link)]. Table S1 shows the list of antibodies used. After incubation with secondary horseradish peroxidase-conjugated antibodies (Bio-Rad), specific proteins were visualized by the enhanced chemiluminescence system using a ChemiDoc™ XRS + imaging system (Bio-Rad).
5
Protein Expression Analysis Protocol
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PPA analysis was performed as described previously [40 (link)], and antibodies were obtained from a number of sources [19 (link)]. The blot was hybridized with secondary horseradish peroxidase-conjugated antibodies (Bio-Rad) and chemiluminescence signal was detected using the ChemiDocXRS System. Differences in protein levels were analyzed by densitometric scanning and normalized using internal standards.
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