HL-60 cells were treated with depicted VAE concentrations for 18 h. Cell lysates were incubated with a human apoptosis array membrane, detecting the relative expression of 35 apoptosis-related proteins per sample according to the manufacturer’s protocol (R&D, Minneapolis, MN, USA). Immunoreactive dots were visualized using Molecular Imager ChemiDoc (Bio-Rad, Germany). Each experiment was carried out in duplicate (n = 2).
Chemidoc molecular imager
Manufactured by Bio-Rad
Sourced in United States, Italy, Germany
The ChemiDoc Molecular Imager is a lab equipment product designed for the capture and analysis of chemiluminescent, fluorescent, and colorimetric signals. It provides high-resolution digital imaging to detect and quantify a variety of biological samples, including protein gels, Western blots, and nucleic acid gels.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (7 mentions)
β actin, by Bio-Rad (6 mentions)
Nitrocellulose membrane, by Bio-Rad (5 mentions)
Image lab analysis software, by Bio-Rad (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (3 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (3 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (3 mentions)
Pre cast polyacrylamide gels, by Bio-Rad (3 mentions)
Pierce ripa buffer, by Thermo Fisher Scientific (3 mentions)
Precast polyacrylamide gel, by Bio-Rad (3 mentions)
Hsp60, by Santa Cruz Biotechnology (3 mentions)
Anti stx7, by Fortis Life Sciences (2 mentions)
Mouse monoclonal anti tuj1, by Fortrea (2 mentions)
Mouse monoclonal anti syb2, by Fortrea (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Western lightning plus ecl kit, by PerkinElmer (2 mentions)
Bradford reagent, by Bio-Rad (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
39 protocols using chemidoc molecular imager
1
Apoptosis Profiling in HL-60 Cells
2
Analyzing Viscum-Induced Cell Signaling
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TC-71 and MHH-ES-1 cells were incubated with viscum, TT or viscumTT in increasing concentrations for 24 h. The cells were washed twice with phosphate-buffered saline and incubated in Lysis Buffer 17 (R&D systems, Minneapolis, MN) containing protease inhibitors (complete Protease Inhibitor Cocktail Tablets, Roche Diagnostics GmbH) to obtain cell lysates. Protein concentration was determined using Bradford solution (Bio-Rad, Munich, Germany). TC-71 and MHH-ES-1 cell lysates (30 μg protein/lane) were separated on SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad system) and blocked with 5% non-fat milk in 50 mM Tris-buffered saline containing 0.05% Tween-20 (TBST) for 1 h at room temperature. Blots were incubated overnight at 4 °C in TBST containing 5% BSA and primary antibody, washed thrice in TBST and incubated 1 h with HRP-conjugated secondary antibodies (anti-rabbit and anti-mouse, Bio-Rad) then visualized by ECL (Thermo Scientific) on a Molecular Imager ChemiDoc (Bio-Rad). Primary antibodies were directed against p-MAPK14 (Thr180/Tyr182, #9211 Cell Signaling Technology, Danvers, MA, USA), LC3B (#2775 Cell Signaling Technology), EIF2AK3 (#3192, Cell Signaling Technology), p-MAPK8 (sc-6254, Santa Cruz biotechnology, Dallas, TX, USA), HSPA5 (#G8918, Sigma-Aldrich) and ß-actin conjugated directly to peroxidase (#A3854, Sigma-Aldrich).
3
Western Blot Analysis of Spinal Cord Samples
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Samples (cells or spinal cord tissue segments at L1–L6) were collected and washed with ice-cold PBS before being lysed in lysis buffer. Then sample lysates were separated by SDS-PAGE and electrophoretically transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 10% whole milk in TBST (Tris-HCl, NaCl, and Tween 20) or 5% bovine serum albumin (BSA) in TBST for 2 h at room temperature, and then probed with primary antibodies at 4°C overnight. After that, bands were detected with horseradish peroxidase (HRP)-coupled secondary antibodies. Data were acquired with the Molecular Imager (ChemiDoc, Bio-Rad) and analyzed with ImageJ software (National Institutes of Health, USA).
4
Measuring PKA Activity in Transfectants
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Stable transfectants were grown for 72 hours, trypsinized, and washed with PBS, 1 × 106 cells were pelleted, and a PKA enzymatic activity assay was performed with a PepTag Assay for Non-Radioactive Detection of cAMP-Dependent Protein Kinase (Promega). Reactions were run in an 0.8% agarose Tris-HCl gel at 100 V for 20 minutes. Images were taken with Bio-Rad Molecular Imager ChemiDoc and Image Lab software.
5
DNA Origami Electrophoresis Analysis
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TriTrack DNA Loading Dye (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added to the DNA origami samples, then subjected to a thermal shock by heating the solution for 3 min at 95°C, followed by cooling on ice. The GeneRuler 1 kb plus DNA ladder (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added to one gel lane. Approximately 200 ng of the DNA was electrophoresed in a 1% agarose gel in TAE buffer (pH 8) at 60 V for ∼3 h. Gels were stained with 0.5 μg/mL ethidium bromide solution for 15 min and rinsed in ddH2O for 15 min. Gel images were generated using the Molecular Imager Chemi Doc (Bio-Rad, Hercules, CA, USA). Images were analysed using ImageJ V2.0. The intensity value of each peak was determined by measuring the distance from the peak apex to the peak base.
6
Western Blotting Protocol for Protein Analysis
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The total cell lysate was obtained by lysing cells in RIPA buffer. To prepare samples for western blot analysis, 30 μg of protein were taken from cell lysates or cytoplasmic fraction solutions and mixed with Laemmli buffer. Afterward, the volumes in all samples were adjusted. To prepare samples with nuclear proteins, Laemmli buffer was added to nuclear pellets obtained by different fractionation methods. Samples (cellular, cytoplasmic, or nuclear) were then heated at 95 °C for 5 min. Afterward, the samples were separated by PAGE (4% stacking gel, 12% resolving gel) and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA) using Mini Trans-Blot cells (Bio-Rad). The membranes were blocked for 40 min in milk (5% solution in TBS) and stained sequentially with primary and secondary antibodies. To obtain the signal, the membranes were treated with ECL Western Blotting Substrate (Promega, Madison, WI, USA) or SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) on a Molecular Imager ChemiDoc (Bio-Rad). If staining with additional antibodies was required, the membranes were incubated in a Restore Western Blot Stripping Buffer (Thermo Scientific) for up to 15 min, washed with TBS solution, blocked with milk, and the staining was repeated.
7
Quantifying Synaptic Vesicle Protein Levels
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Protein samples were separated on SDS-polyacrylamide mini-gels and blotted onto PVDF membranes (Millipore). As primary antibodies, rabbit polyclonal anti-Stx7 (Bethyl Laboratories), mouse monoclonal anti-Syb2 (Cl69.1), and mouse monoclonal anti-Tuj1 (Covance) were used. Blots were further incubated with secondary antibodies (anti-mouse IgG or anti-rabbit IgG, respectively) coupled to horseradish-peroxidase and developed using a Western Lightning Plus-ECL kit (PerkinElmer). Signals were detected using a Molecular Imager ChemiDoc (BioRad).
To estimate the copy number of Stx7 per SV, the CPG-purified SV fraction (a gift from Reinhard Jahn) with known protein concentration and vesicle number, was subjected to western blotting together with various amounts of standard proteins for Stx7 and Syb2. Band intensities in acquired images were quantified using Quantity One software (BioRad).
To estimate the copy number of Stx7 per SV, the CPG-purified SV fraction (a gift from Reinhard Jahn) with known protein concentration and vesicle number, was subjected to western blotting together with various amounts of standard proteins for Stx7 and Syb2. Band intensities in acquired images were quantified using Quantity One software (BioRad).
8
Western Blot Analysis of Brain Endothelial Cells
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Sorted brain CD31+ endothelial cells were washed with PBS and cell pellets were lysed with Radioimmunoprecipitation assay (RIPA) buffer (Serva) with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific) and an equal volume of SDS-containing sample buffer (Sigma-Aldrich) was added and boiled. Total proteins were separated on a 4 to 15% SDS-polyacrylamide gel and transferred onto a nitrocellulose membrane (Bio-Rad). After blocking with 5% nonfat dry milk in TBS-T (Tris-buffered saline with 0.1% Tween 20), immunoblots were incubated with primary antibodies overnight, washed in TBS-T, further incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (Advansta) for 1 h, and visualized with chemiluminescence (Clarity Western ECL substrate, Bio-Rad) on a Chemidoc MP imaging system (Bio-Rad). The antibodies used for immunoblot analysis were against ZO-1 (Thermo Fisher Scientific), Claudin-5 (Sigma-Aldrich), and β-actin-HRP (Biolegend). The densitometric analysis of each protein signal was obtained using the Molecular Imager Chemidoc (Bio-Rad) and normalized against β-actin expression.
9
Protein Extraction and Western Blot
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After VAE incubation, HL-60 cells were washed twice with PBS and lysed in RIPA buffer containing protease inhibitors. Protein lysates were cleared of cellular debris by centrifugation for 10 min at 13 000 rpm at 4°C. The protein concentration was determined using Bradford Reagent (Bio-Rad, Munich, Germany). Equivalent amounts of proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, Munich, Germany). Primary antibody incubations were performed overnight at 4°C. Detection was performed with HRP-conjugated secondary antibodies (Bio-Rad, Munich, Germany) which were visualized by ECL (Thermo Fisher Scientific, Bonn, Germany) and Molecular Imager ChemiDoc (Bio-Rad, Munich, Germany).
10
Western Blot Analysis of Endol Protein
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Cell lysates were electrophoresed on 10% SDS polyacrylamide gels and transferred to PVDF membranes (Millipore) for 30 min with a semi-dry blotting device (BioRad). Membranes were blocked for 30 min at RT with blocking buffer (5% skim milk in Tris-buffered saline (pH 7.4) supplemented with 0.05% Tween 20 (TBS-T)). Primary antibodies were used at the following dilutions: anti-EndoI (1:1,000), and anti-β-actin (1:1,000). Secondary antibodies conjugated with HRP were used at 1:10,000. Chemiluminescence signals of the HRP enzymatic reaction were detected with Western Lightning Plus-ECL (PerkinElmer) using a Molecular Imager ChemiDoc (BioRad). Signal intensities of bands were quantified using a Gel tool in Fiji software.
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