Contracting atrial muscle strips from mice and human hearts that were utilised in the atrial organ bath experiments were rapidly brought to the temperature of liquid nitrogen (Gergs et al. 2021d (link)). The samples were kept at – 80 °C and were then subjected to biochemical analysis. Western blotting for serine 16 phosphorylated PLB and the use of calsequestrin as the loading control have been described in a previous study (e.g. Neumann et al. 2021a (link), 2022 (link)). The following primary antibodies were used: anti-serine 16-phosphorylated PLB (anti-PLB-Ser16; 1:5000; Badrilla, Leeds, UK, Cat. # A010-12AP) and anti-calsequestrin (1:20,000; Abcam, Cambridge, UK, Cat. # ab3516). Visualisation of the signal was performed using Immobilon Western chemiluminescent horseradish peroxidase substrate (Merck Millipore, Darmstadt, Germany). We homogenised the cardiac muscle strips, measured the protein concentration in the muscle homogenates, subjected the homogenates to electrophoresis, transferred them to nitrocellulose membranes, incubated the membranes with primary and secondary antibodies and then quantified the results in line with our lab procedures, which have been outlined in several published studies (Gergs et al. 2010 (link), 2019a (link), b (link); Boknik et al. 2018 (link), 2019 (link)).
Immobilon western chemiluminescent horseradish peroxidase substrate
Manufactured by Merck Group
Sourced in United States, Germany, China
Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate is a laboratory reagent used to detect and quantify protein levels in Western blot analysis. It is a chemiluminescent substrate that reacts with horseradish peroxidase (HRP) enzyme to produce a light signal, which can then be captured and measured.
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Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Polyvinylidene difluoride membrane, by Merck Group (5 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
Image lab software, by Bio-Rad (3 mentions)
Chemidoc xrs system, by Bio-Rad (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Vinculin, by Merck Group (2 mentions)
Anti tubulin b512, by Merck Group (2 mentions)
Anti cleaved caspase 3 9661, by Cell Signaling Technology (2 mentions)
Complete protease inhibitor mixture, by Roche (2 mentions)
Any kd mini protean tgx precast gel, by Bio-Rad (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by GE Healthcare (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ripa lysis kit, by Applygen (2 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
Polyvinylidene fluoride microporous membrane, by Merck Group (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Ibright imaging system, by Thermo Fisher Scientific (2 mentions)
46 protocols using immobilon western chemiluminescent horseradish peroxidase substrate
1
Quantifying Phosphorylated Phospholamban in Cardiac Muscle
2
Immunoblotting of Brain Membrane Proteins
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The lysates of the light membrane fraction of mouse brain and the immunoprecipitated samples were mixed with the SDS sample buffer and boiled for 5 min. The samples were then separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Merck). After being blocked with 5% skim milk in Tris buffered saline (TBS), 20 mM Tris–HCl (pH 7.4) and 150 mM NaCl with 0.05% Tween 20 (TBS-T), the membranes were incubated with the indicated Abs. After being washed with TBS-T three times, the membranes were incubated with the horseradish peroxidase-conjugated rabbit or mouse IgG pAb (Jackson ImmunoResearch Laboratories). The signals for the proteins were detected using Immobilon Western Chemiluminescent horseradish peroxidase Substrate (Merck).
3
Western Blot Analysis of Protein Expression
4
Protein Analysis of Nitrogenous Compound Effects
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U251/MCF-7 cells (2×105/well) were cultured in 6-well plates in the presence of the nitrogenous heterocyclic compound (20 and 50 μM) at 37°C for 72 h. The cells were lysed, and the total proteins were extracted by centrifugation at 12,000 g at 4°C for 10 min using protein extraction buffer (Bioo Scientific, Austin, TX, USA). Protein was measured using the Bradford assay (Beyotime Biotechnology, Shanghai, China), and 50 ng of the protein was separated via 12% SDS-PAGE and then transferred onto a polyvinylidene fluoride membrane. The membrane was blocked using 1% BSA at 37°C for 1 h. The membrane was incubated with AGPS (1:2,000, sc-374201), p21 (1:1,500; sc-166630), p27 (1:1,500; sc-71813), Bcl-2 (1:1,500; sc-23960), survivin (1:1,500; sc-101433) and Bim (1:1,500; sc-374358) antibodies at 4°C overnight and was then incubated at 37°C for 1 h with peroxidase-labeled anti-rabbit immunoglobulin G (1:2,000). The membrane was washed three times with PBS containing 0.05% Tween20. The membrane was visualized using Immobilon Western chemiluminescent horseradish peroxidase substrate (EMD Millipore, Billerica, MA, USA). β-Actin (1:5,000; sc-58673) was used as the control. All antibodies were purchased from Santa Cruz (Dallas, TX, USA).
5
Western Blot Analysis of ER Stress Markers
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Proteins were extracted from intestinal mucosal tissues using cell lysis buffer (P0013; Beyotime, Shanghai, China), and the protein concentration was assessed with BCA Protein Assay Kit (P0011; Beyotime). Then proteins were run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels and transferred to Polyvinylidene Fluoride membranes (IPVH00010; Merck Millipore, Billerica, MA). Membranes were blocked with 5% milk at room temperature for 1 h. Then membranes were incubated with primary antibodies at 4°C overnight. Primary antibodies included anti-PDIA3 antibody (1:1,000, ab13506; Abcam, Cambridge, MA), anti-Bip antibody (1:1,000, 3177S; CST, Cambridge, MA), anti–caspase 12 antibody (1:1,000, sc-21,747; Santa Cruz, Dallas, TX), anti-Chop antibody (1:1,000, 2895S; CST), anticleaved caspase 3 antibody (1:1,000, 9664S, CST), and anti–β-actin antibody (1:5000, 66,009-1-Ig; Proteintech, Rosemont, IL). After incubating membranes with secondary antibodies at room temperature for 1 h, membranes were incubated with Immobilon Western chemiluminescent horseradish peroxidase substrate (WBKLS0500; Merck Millipore). Blot signals were detected with ImageQuant LAS 500 imager (General Electric, Boston, MA) and analyzed with ImageJ software (National Institutes of Health, Bethesda, MD).
6
Western Blot Analysis of ALDH2 in Hepatic Cells
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Hepatic whole-cell lysates were prepared as described previuosly (49 (link)). The denatured protein samples (20 μg) were fractionated on Any kD Mini-PROTEAN TGX Precast Gels (Bio-Rad Laboratories, Hercules, CA). Primary antibodies and the titers used for western blotting were as follows: goat polyclonal anti-ALDH2 (N-14) (sc-48838; Santa Cruz Biotechnology, Santa Cruz, CA;1:1000); rabbit monoclonal anti-β-actin (13E5; Cell Signaling Technology; 1:2000). These were then reacted with the appropriate horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Little Chalfont, UK). The signal was visualized using an Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate (Merck Millipore, Darmstadt, Germany) and was exposed using a ChemiDoc XRS system equipped with Image Lab software (Bio-Rad Laboratories).
7
Western Blot Analysis of Survivin in Tumor Tissues
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Tumor tissues obtained from mice in each experimental group were homogenized in cold lysis buffer with 1 mM phenylmethanesulfonyl fluoride and subsequently lysed for 60 minutes on ice. Protein concentrations were determined via Coomassie Blue Fast Staining (Beyotime Institute of Biotechnology, Shanghai, China). Protein samples (50 μg) from each experimental group were separated via 10% SDS-PAGE and subsequently transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). Following this, membranes were blocked using Tris-buffered saline containing Tween 20 and 5% skimmed milk for 3 hours and subsequently incubated with the survivin antibodies (1:750) overnight at 4°C. GAPDH antibodies (1:15,000) were used as an endogenous reference. Following this, membranes were incubated with secondary antibodies (1:4,000) at room temperature for 1 hour. The blots were then visualized using Immobilon™ Western Chemiluminescent Horseradish Peroxidase Substrate (EMD Millipore) and the Tanon-4500 Gel Imaging System and subsequently quantified using GIS ID Analysis Software v4.1.5 (Tanon Science & Technology Co., Ltd., Shanghai, China).
8
Western Blot Analysis of Protein Markers
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Cells grown in culture were rinsed with ice-cold phosphate-buffered saline (PBS), scraped, and lysed with 1× EBC buffer (50 mM Tris [pH 8.0], 120 mM NaCl, 0.5% NP-40) supplemented with complete protease inhibitor mixture (Hoffman-La Roche Ltd., Basel, Switzerland). Equal amounts of protein extract, as determined by the Bradford method, were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted onto polyvinylidene difluoride membranes. After blocking in Tris-buffered saline with 5% nonfat dry milk, the membranes were probed with the following primary antibodies: anti-HA (HA-11; Covance Research Products Inc., Denver, CO, USA), anti-HIF-1α (NB100-479; Novus International Inc, St Louis, MO, USA), anti-cleaved Caspase 3 (9661, Cell Signaling Technology, Inc, Danvers, MA, USA), anti-p53 (Calbiochem, Darmstadt, Germany), vinculin (Sigma-Aldrich), and anti-tubulin (B-512, Sigma-Aldrich). Horseradish-peroxidase-conjugated secondary antibodies were from Pierce, and immobilon Western chemiluminescent horseradish peroxidase substrate was purchased from EMD Millipore, Billerica, MA, USA.
9
Western Blot Analysis of Oxidative Stress Markers
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Aliquots of protein (20–30 μg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Temecula, CA, USA). The blots were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 and were then incubated with the antibodies. The primary antibodies used were anti-Prdx-SO2/3 (ab16830; Abcam, Cambridge, UK), anti-SOD1 [25 (link)], anti-Prdx1 [25 (link)], anti-Prdx2 (LF-PA0091; AbFrontier, Seoul, Korea), anti-Prdx3 (LF-MA0044; AbFrontier), anti-Prdx4 [17 (link)], anti-endoplasmic reticulum stress-C/EBP homologous protein (CHOP) (sc-7351; Santa Cruz Biotechnology, CA, USA), anti-PDI (sc-20132; Santa Cruz Biotechnology), anti-IRE1α (number 3294; Cell Signaling Technology, Danvers, MA, USA), anticleaved caspase-3 (number 9664; Cell Signaling Technology), and anti β-actin (sc-69879; Santa Cruz Biotechnology). Horseradish peroxidase-conjugated goat anti-rabbit, anti-mouse, and anti-guinea pig IgG antibodies (Santa Cruz Biotechnology) were used as the secondary antibodies. After washing, immune reactive bands were detected by measuring the chemiluminescence using Immobilon Western chemiluminescent horseradish peroxidase substrate (EMD Millipore, Temecula, CA, USA) on an image analyzer (ImageQuant LAS 500; GE Healthcare Life Sciences, Buckinghamshire, UK).
10
Western Blot Analysis of Protein Samples
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Whole cell lysates were prepared as described45 (link). The denatured protein samples (20 μg) were fractionated on Any kD Mini-PROTEAN TGX Precast Gels (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Primary antibodies and the titers used for western blotting were as follows: goat polyclonal anti-ALDH2 (N-14) (sc-48838; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; 1:1000); mouse monoclonal anti-FLAG M2 (F1805; Sigma-Aldrich; 1:1000); rabbit monoclonal anti-β-actin (13E5; Cell Signaling Technology, Inc.; 1:2000). These were then reacted with the appropriate horseradish peroxidase-conjugated secondary antibody (GE Healthcare, Little Chalfont, UK). The signal was visualized using an Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate (Merck Millipore, Darmstadt, Germany) and was exposed using a ChemiDoc XRS system equipped with Image Lab software (Bio-Rad Laboratories, Inc.).
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