Cell lysates for Western blot were obtained by incubating HEK293 (for uEV Western blot) and PC3 or DU145 (for cEV Western blot) cell pellets at a concentration of 2 × 10 (Hoshino et al., 2020 (link)) cells in 25 ul of lysis buffer (50 mM Tris, pH 7.5, 0.15 M NaCl, 1% Triton X‐100) with 2% complete protease inhibitor (Roche) for 20 min on ice, followed by an 18,516×g centrifugation for 15 min at 4 °C to recover the supernatant. Both cell lysates and EV pellets (10 (Spinelli et al., 2021 (link)) particles) were mixed with Laemmli sample buffer (BioRad), without reducing agent. After boiling for 5 min at 95 °C, samples were loaded on a 4–15% Miniprotean TGX stain‐free gels (BioRad). Total proteins were imaged from the stain‐free gels with the ChemiDoc Touch Imager (BioRad). Transfer was performed on Immuno–Blot PVDF membranes (BioRad), with the Trans–Blot Turbo Transfer System (BioRad) during 7 min. Blocking was performed during 30 min with Blocking Reagent (Roche) in TBS 0.1% Tween. Primary antibodies were incubated overnight at 4 °C and secondary antibodies during 1 h at room temperature (RT). Development was performed using either the Clarity Western ECL Substrate (BioRad) or the Immobilon Forte Western HRP substrate (Millipore), and the ChemiDoc Touch Imager (BioRad). Intensity of the bands was quantified using ImageJ.
Chemidoc touch imager
Manufactured by Bio-Rad
Sourced in United States, Sweden
The ChemiDoc Touch Imager is a laboratory instrument designed for the detection and analysis of chemiluminescent, fluorescent, and colorimetric signals. It provides high-quality imaging and quantitative analysis capabilities for a variety of life science applications.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software, by Bio-Rad (13 mentions)
Laemmli sample buffer, by Bio-Rad (5 mentions)
Clarity western ecl substrate, by Bio-Rad (5 mentions)
Ripa buffer, by Merck Group (5 mentions)
Immuno blot pvdf membrane, by Bio-Rad (4 mentions)
Immobilon forte western hrp substrate, by Merck Group (4 mentions)
Trans blot turbo transfer system, by Bio-Rad (4 mentions)
Image lab 5, by Bio-Rad (4 mentions)
Iblot2, by Thermo Fisher Scientific (4 mentions)
Hyglo quick spray, by Thomas Scientific (4 mentions)
Blocking reagent, by Roche (3 mentions)
Mini protean tgx stain free gel, by Bio-Rad (3 mentions)
Trans blot turbo, by Bio-Rad (3 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Promega (3 mentions)
Phosstop, by Roche (3 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Horseradish peroxidase conjugated goat anti mouse or goat anti rabbit igg, by Merck Group (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (3 mentions)
63 protocols using chemidoc touch imager
1
Extracellular Vesicle Protein Analysis
2
Density-based Protein Separation and Analysis
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Densities of the 1-ml fractions from the flotation centrifugation analyses were determined from three independent experiments by measuring the weight of three 100 µl subfractions from each fraction. The fractions were precipitated with trichloroacetic acid (TCA) and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE gels were stained with Coomassie, Sudan black, or both and imaged using a ChemiDoc Touch imager (BioRad) or used for Western blotting. Polyclonal antibodies (pAb) against P12 (1:13,333; GeneCust) and phi6 (1:2500) and a monoclonal antibody against GFP (1:25,000; ab291, Abcam) were used as primary antibodies. HRP-labeled anti-rabbit pAb (1:125,000; Sigma Aldrich) or anti-mouse pAb (1:5000; PI-2000; Vector Laboratories Inc.) were used as secondary antibodies. Chemiluminescence was detected using a Western Lightning ECL Kit (Perkin-Elmer) and ChemiDoc Touch imager (BioRad) with 600 s of exposure time. Mass spectrometric analyses of P12 were performed in the Proteomics Unit of the University of Helsinki using liquid-chromatography–mass spectrometry × 2 (LC–MS/MS). Quantitative analyses of the EM thin sections was done using Aida Image Analyzer v 4.5.
3
Western Blot Analysis of Cell Lysates and EVs
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Cell lysates (CL) for Western blot were obtained by incubating cell pellets at a concentration of 1 × 106 cells in 25 uL of lysis buffer (50 mM Tris, pH 7.5, 0.15 M NaCl, 1% Triton X-100) with 2% complete protease inhibitor (Roche) for 20 min on ice, followed by a 18,516×g centrifugation for 15 min at 4 °C to recover the supernatant. EVs from 20 × 106 cells (or specified number of particles) and CL from 0.2 × 106 cells were mixed with Laemmli sample buffer (BioRad), without reducing agent. After boiling for 5 min at 95 °C, samples were loaded on a 4–15% Mini-protean TGX stain-free gels (BioRad). Total proteins were imaged from the stain-free gels with the ChemiDoc Touch Imager (BioRad). Transfer was performed on Immuno-Blot PVDF membranes (BioRad), with the Trans-Blot Turbo Transfer System (BioRad) during 7 min. Blocking was performed during 30 min with Blocking Reagent (Roche) in TBS 0.1% Tween for most antibodies or with 5% milk in PBS 0.1% Tween for the anti-GFP antibody. Primary antibodies were incubated overnight at 4 °C and secondary antibodies during 1 h at room temperature (RT). Development was performed using either the BM Chemiluminescence Western blotting Substrate (POD) (Roche), Clarity Western ECL Substrate (BioRad) or the Immobilon Forte Western HRP substrate (Millipore), and the ChemiDoc Touch Imager (BioRad). Intensity of the bands was quantified using ImageJ.
4
Western Blot Analysis of Cell Lysates and EVs
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Cell lysates for Western blot were obtained by incubating HEK293 cell pellets at a concentration of 1 × 10 6 cells in 25 uL of lysis buffer (50 mM Tris, pH 7.5, 0.15 M NaCl, 1% Triton X-100) with 2% complete protease inhibitor (Roche) for 20 min on ice, followed by a 18,516×g centrifugation for 15 min at 4 °C to recover the supernatant. Both cell lysates and EV pellets from 5ml urines were mixed with Laemmli sample buffer (BioRad), without reducing agent. After boiling for 5 min at 95 °C, samples were loaded on a 4-15% Miniprotean TGX stain-free gels (BioRad). Total proteins were imaged from the stain-free gels with the ChemiDoc Touch Imager (BioRad). Transfer was performed on Immuno-Blot PVDF membranes (BioRad), with the Trans-Blot Turbo Transfer System (BioRad) during 7 min. Blocking was performed during 30 min with Blocking Reagent (Roche) in TBS 0.1% Tween. Primary antibodies were incubated overnight at 4 °C and secondary antibodies during 1 h at room temperature (RT). Development was performed using either the Clarity Western ECL Substrate (BioRad) or the Immobilon Forte Western HRP substrate (Millipore), and the ChemiDoc Touch Imager (BioRad). Intensity of the bands was quantified using ImageJ.
5
Soft Agar Colony Growth Assay
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Soft agar colony growth assay was performed as described previously [67 (link)]. 20,000 cells were plated per well in a 6-well plate in 1 ml of 0.1% agarose in media on top of a 2-ml bottom layer of 0.5% agarose in media. Plates were incubated at 37°C in incubator for one week. Cells were fed every other day and images were taken using ChemiDoc Touch Imager (Bio-Rad) or CL1500 Imaging System (Thermo Fisher Scientific). Colony area was measured using the ImageJ software. Each experiment contained at least three replicates per condition, and every experiment was performed at least three times.
6
SDS-PAGE and Immunoblotting for Protein Analysis
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Samples were mixed with Laemmli sample buffer (Laemmli, 1970 ) and boiled for 3 min. Proteins were electrophoretically separated using SDS–PAGE, transferred to nitrocellulose using a Transblot Turbo (BioRad, Hercules, CA), and immunoblotted with the indicated antibodies. HRP-conjugated secondary antibodies were used at 5 ng/ml, and immunoreactivity was detected with enhanced chemiluminescence (PerkinElmer, Waltham, MA) using the ChemiDoc Touch Imager (BioRad). Relative protein levels were determined by densitometric analysis of immunoreactive bands using the ChemiDoc imaging software or ImageJ software (National Institutes of Health).
7
Western Blot Protocol for Protein Analysis
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Cells were lysed with RIPA buffer (Sigma) supplemented with protease inhibitor cocktail (Promega) and PhosSTOP (Roche) and placed on ice for 30 min, with vortexing every 10 min. Lysates were pulse-sonicated for 30 sec, with 10-s burst-cooling cycles, at 4°C, boiled in reducing sample buffer for 5 min and resolved on 4–12% Bis-Tris Bolt gels and transferred using an iBlot 2 (Thermo Fisher). Blots were blocked in 2.5% milk in 1% TBS-Tween before staining with antibodies (Table S5 ). The signals were detected with HyGLO Quick Spray (Denville Scientific) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher) and viewed on a ChemiDoc Touch Imager with Image Lab 5.2 software (BioRad).
8
Quantitative Western Blot Analysis
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Cell lysates were prepared using RIPA buffer (150 mM NaCl; 50 mM Tris–HCl pH 8.0; 0.5%NP-40; 1 mM PMSF, protease inhibitor cocktail). After protein quantification by BCA assay (ThermoFischer, United States), 20 ug of Laemli-diluted cell lysates were loaded on 17% SDS-PAGE, run on TRIS-Glycine running buffer, followed by transfer to a nitrocellulose membrane (Bio-RAD, United States). Following 1 h blocking in PBST-diluted 5% nonfat milk, membranes were incubated with primary antibodies: LC3B (1:1,000, #2775S, Сell Signaling, United States), p62 (1:1,000, #5114, Сell Signaling, United States) or β-actin (1:5000, A3854, Sigma-Aldrich, United States). After PBST washing, secondary anti-mouse or anti-rabbit antibodies (1:10,000; Sigma-Aldrich, United States) conjugated with horseradish peroxidase were used. ECL system (ThermoScientific, United States) and ChemiDoc Touch Imager (Bio-Rad, United States) were applied for detection.
9
Western Blot Analysis of Frozen Tissues
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Fresh frozen tissue lysis and Western blotting (WB) were performed as previously described [14 (link)]. The primary antibodies are provided in Additional file 1 : Table S1, and many were previously tested in autopsy tissue [14 (link)]. WBs for each antibody were repeated at least twice, with similar results. The densitometric analysis was performed by scanning the X-ray films with optimal exposures on a ChemiDoc™ Touch imager (Bio-Rad, Hercules, CA). The bands were quantified by using Image Lab 6.0 software (Bio-Rad). Individual protein values were normalized to the corresponding actin or IDH1-R132H values, except for phosphoprotein values that were normalized to the corresponding unphosphorylated protein values. Minus values were manually adjusted as zero. Results were expressed as percent of the highest normalized values.
10
Western Blot Protein Extraction and Detection
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Cells grown on tissue culture plates were washed with 1X PBS and prepared in cold Triton lysis buffer (1% Triton X-100, 40 mM HEPES (pH 7.5), 120 mM sodium chloride, 1 mM EDTA, 1 mM phenyl methylsulfonyl fluoride, 10 mM sodium pyrophosphate, 1 μg/ml each of cymostatin, leupeptin and pepstatin, 10 μg/ml each of aprotinin and benzamidine, 2 μg/ml antipain, 1 mM sodium orthovanadate, 50 mM sodium fluoride). For immunoblotting, cell lysates were centrifuged at 14,000 rpm for 3 min at 4°C to remove cell debris. Protein concentration was determined using the Bio-Rad Protein Assay (Bio-Rad) with bovine serum albumin (RMBIO) as standard then were prepared in Laemmli SDS-PAGE sample buffer. Aliquots of protein lysate were resolved by SDS-PAGE, transferred to nitrocellulose membranes (0.45 μm) (Bio-Rad, Hercules, CA) and immunoblotted with the indicated antibodies at 1:1000 dilution, followed by horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG (Sigma-Aldrich) and Amersham ECL Select Western Blotting Detection Reagent or Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Hudson, NH). Signals were detected using ChemiDoc Touch Imager (Bio-Rad) or CL1500 Imaging System (Thermo Fisher Scientific). For western blot signal quantitation, the Image Lab software (Bio-Rad) was used.
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