Tumors from all of the mice were excised, and tumor tissues were homogenized in radioimmunoprecipitation assay lysis buffer containing protease and phosphatase inhibitors. The tissue lysate was then centrifuged at 15,000 rpm, and the supernatant was obtained, followed by protein quantification using the Pierce BCA Protein Assay Kit. An equal amount of 40 μg of protein lysates was mixed with 1× Laemmli SDS sample buffer (Alfa Aesar) and electrophoresed at a constant of 200 V using 10% polyacrylamide gel. The samples were then transferred to polyvinylidene difluoride (PVDF) membrane at a constant current of 35 mA, followed by blocking in tris-buffered saline and 0.1% Tween 20 (TBST) containing 5% skim milk. PVDF membranes were incubated in 1% BSA in TBST with GrB (1:1000 dilution), cleaved caspase-3 (1:1000 dilution), and β-actin (1:2000 dilution) at 4°C for overnight. Appropriate washes with TBST were performed to remove excess antibodies, and membranes were incubated with horseradish peroxidase–conjugated anti-rabbit antibody (1:5000 dilution) in 1% BSA in TBST for 1 hour at room temperature. Bio-Rad’s Clarity ECL was used for band detection, and image analysis and quantification were done using Image Lab 6.0.1 and ImageJ 2.0.0.
Clarity ecl
Manufactured by Bio-Rad
Sourced in United States, United Kingdom, Canada
The Clarity ECL is a chemiluminescent detection system designed for Western blot analysis. It provides a sensitive and reliable method for detecting proteins on membranes using a luminescent substrate reaction.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc mp imaging system, by Bio-Rad (15 mentions)
Image lab software, by Bio-Rad (13 mentions)
Nitrocellulose membrane, by Bio-Rad (13 mentions)
Chemidoc, by Bio-Rad (12 mentions)
Trans blot turbo system, by Bio-Rad (12 mentions)
Pierce ecl, by Thermo Fisher Scientific (12 mentions)
Trans blot turbo, by Bio-Rad (10 mentions)
Chemidoc touch imaging system, by Bio-Rad (9 mentions)
Pvdf membrane, by Bio-Rad (9 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Chemidoc imaging system, by Bio-Rad (7 mentions)
Chemidoc mp, by Bio-Rad (6 mentions)
Laemmli buffer, by Bio-Rad (6 mentions)
β actin, by Cell Signaling Technology (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Gapdh, by Cell Signaling Technology (5 mentions)
Ripa buffer, by Merck Group (5 mentions)
Image lab 5, by Bio-Rad (4 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
155 protocols using clarity ecl
1
Immunoblotting analysis of tumor lysates
2
Western Blot Protein Detection Protocol
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Cells were lysed in a lysis buffer comprised of 50 mM Tris-HCl pH 7.5, 150 mM sodium chloride, 1% Triton-X-100 and prepared in 3x Laemmli sample buffer. Following the separation of proteins on 7.5% SDS-PAGE gels, they were transferred to polyvinylidene difluoride (PVDF) membranes pre-wetted in methanol. Membranes were blocked in 10% non-fat milk powder dissolved in either phosphate-buffered saline containing 0.15% Trition-X-100 (rabbit anti-CHT) or Tris-buffered saline containing 0.1% Tween-20 (rabbit anti-actin, pan/phospho-PKB/Akt), then incubated overnight at 4°C with antibody in 5% milk powder in the appropriate buffer. After washes, membranes were incubated for 1–2 h at room temperature with peroxidase-conjugated goat anti-rabbit secondary antibody in 5% milk powder, then washed again. Immunoreactive proteins were detected using Enhanced ChemiLuminescence (Amersham ECL, GE Health Sciences, QC, Canada or Clarity ECL, BioRad, Mississauga, ON, Canada) and imaged with a ChemiDoc MP Imaging system (BioRad). Quantification was performed using the BioRad software ImageLab5.
3
Western Blot Analysis of Phosphorylated NF-κB
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Cell pellets were lysed with lysis buffer (1 M HEPES, 0.5 M NaF, 0.5 M EGTA, 2.5 M NaCl, 1 M MgCl2, 10% glyercol, 1% Triton X‐100) with PhosSTOP phosphatase inhibitor (Roche, Basel, Switzerland). Protein concentrations were measured with a Bradford assay (Sigma-Aldrich). Samples (10 μg protein) were separated on a 10% polyacrylamide SDS-PAGE in Tris-HCl buffer then transferred onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked in 2.5% bovine serum albumin (BSA; BioShop Canada Inc.) in Tris-buffer saline (TBS) with 0.1% Tween (BioShop Canada Inc.) (TBST) for 2 hours. Membranes were subsequently probed with anti-phospho-NF-κBp65 (1:1000) (New England Biolabs, Whitby, ON) and anti-pan-NF-κBp65 (1:300) (Santa Cruz Biotechnologies, Dallas, TX) in 2.5% BSA (BioShop Canada Inc.) and mouse anti-rabbit IgG-HRP secondary antibody (1:10,000) in 2.5% BSA (BioShop Canada Inc.). Immunoblots were visualized using Clarity ECL (Bio-Rad Laboratories) on the Alpha Innotech HD2 imager using HD2 Imaging software (Thermo Fisher Scientific) for imaging. Full membrane images are provided (Supplementary Figure 1 ).
4
Western Blotting Protocol for Protein Analysis
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Western blots were performed as previously described (16). Briefly, cells were lysed for 15 min. on ice in RIPA lysis buffer with cOmplete protease inhibitors (Promega) and phosphatase inhibitor cocktail 3 (Sigma). Insoluble material was pelleted out, and 1 µg/µL samples were made and run on PROTEAN TGX SDS-PAGE gels (Bio-rad) and transferred onto PVDF membranes, and blocked with 5% nonfat dry milk for 1 hour at room temperature. Primary antibodies were added overnight at 4 degrees celcius, then washed with TBST. HRP-conjugated secondary antibodies (Promega) were added for 1 hour at room temperature at 1:10,000 dilution. Membranes were then washed with TBST, and visualized using Clarity ECL (BioRad) on a ChemiDoc system (BioRad).
5
Evaluating PEDV mAb Reactivity by Western Blot
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The reactivity of PEDV mAbs developed here were evaluated by Western blots. Each antibody was tested against sucrose purified whole virus preparations, a recombinant/truncated version of the S protein (aa 630–800; Hain et al., 2016 (link)), and a recombinant N protein (Okda et al., 2015 (link)). Approximately, 20 µg of each antigen were resolved by SDS-PAGE in 4–20% acrylamide gels (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membranes. Blots were incubated with 5% non-fat-dry-milk tris buffered saline (TBS)−0.1% Tween 20 (TBS-T) solution for 1 h at RT and probed with the MAbs and control antibodies overnight at 4 °C. Blots were washed three times with TBS-T for 10 min at RT and incubated with a goat anti-mouse IgG-HRP- or goat anti-mouse IgM-HRP-conjugated antibody for 2 h at RT. Blots were washed three times with TBS-T for 10 min and developed by using a chemiluminescent substrate (Clarity, ECL; Bio-Rad, Hercules, CA).
6
Western Blot Analysis of Synaptic Proteins
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Western blots were performed with Mini-PROTEAN® TGX™ Precast Gels from Bio-Rad (California 94547 USA).gradient 4–15% Gels were transferred on Nitrocellulose blotting membrane (GE Healthcare Life Sciences) using Towbin buffer (25 mM Tris, 192 mM Glycine, 20% Methanol). Membranes were blocked with Tris-buffered saline TBS (50 mM Tris p < h 7–150 mM NaCl) with 5% Milk for 1 h at room temperature, incubated with primary and secondary antibodies as indicated below, and then developed with Bio-Rad's Clarity ECL on ChemiDoc Touch Imaging System (Biorad). For western blot of crude synaptosomal proteins, 30 ug of proteins was used.
7
Immunoblot Analysis of Taste Receptors
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For endogenous T1R1 and T1R3 immunoblot visualization, cell pellets were resuspended in RIPA Buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% IGEPAL CA-630, 1% deoxycholate, 1 mM DTT, DNase, and Roche cOmplete Protease Inhibitor Cocktail) and lysed via sonicating water bath. Protein concentrations of post 800× g lysates were estimated by Bradford DC Assay (BioRad Laboratories, Hercules, CA, USA) then 60 µg protein/lane was loaded into a 4-12% Bis-Tris gel. After electrophoresis, the resulting gel was transferred to nitrocellulose and the membrane was blocked in 5% milk in Tris-Tween (50 mM Tris, 150 mM NaCl, and 0.025% Tween-20) for at least 1 h. The primary antibody was diluted 1:1000 in Tris-Tween containing 5% bovine serum albumin and incubated for at least 2 h. The secondary antibody goat anti-rabbit IgG-horseradish peroxidase was diluted 1:1000 in Tris-Tween containing 5% milk. After extensive washing, blots were incubated in Clarity ECL (BioRad Laboratories, Hercules, CA, USA) as per manufacturers’ protocol, and resulting immunoblots were visualized using a ChemiDoc MP Imaging System (BioRad Laboratories, Hercules, CA, USA). Final images were processed using Image Lab Software (BioRad Laboratories, Hercules, CA, USA).
8
Protein Extraction and Analysis
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Cells were lysed in RIPA buffer (R0278, Sigma) supplemented with protease and phosphatase inhibitor cocktails (Roche), then homogenised by passing the lysate through 21G needle and syringe. Lysates were clarified by centrifugation and total protein content analysed by BCA assay. For analysis of SDC3 protein levels, 100 μg of total protein was precipitated with methanol overnight at − 20 °C then centrifuged at 13,000 × g to pellet. Pellets were washed once with ice-cold acetone and resuspended in heparinase buffer (100 mM sodium acetate, 0.1 mM calcium acetate). Glycosaminoglycan chains were removed using 0.5 mU heparinase III (Ibex, Canada) and 0.5 mU chondroitinase ABC (Sigma) for 4 h at 37 °C. Total protein 20 µg (or 40 µg for SDC3 analysis) was separated by SDS-PAGE and transferred onto nitrocellulose membranes (Hybond, GE Healthcare). Primary antibodies used were: p-AKT (1:3000, #4060, CST), AKT (1:3000, #9272, CST), p-ERK (1:3000, #9101, CST), ERK (1:3000, #9102, CST), p-Tyrosine (1:2000, #8954, CST), GAPDH (1:8000, G8795, Sigma) and SDC3 (0.14 µg/ml, AF2734, R&D). Secondary HRP-conjugated antibodies were used at 1:10,000 (Santa Cruz). Membranes were visualised using chemiluminescence (Clarity ECL, Bio-Rad) and imaged using ImageQuant-Las4000 (GE Healthcare). Band intensity was analysed using ImageJ.
9
AKT Phosphorylation in Offspring Liver
10
Western Blot Analysis of Cellular Proteins
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Cells (2 × 106) were plated in six‐well plates and, after appropriate stimulations, they were lysed using radioimmunoprecipitation assay buffer (50 mmol L−1 Tris–HCl, 150 mmol L−1 NaCl, 1 mmol L−1 ethylenediaminetetraacetic acid, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) supplemented with cOmplete EDTA‐free protease inhibitor cocktail (Sigma) and PhosSTOP phosphatase inhibitor tablets (Sigma). Cell lysates (10 µg protein measured by bicinchoninic acid assay; Thermo Fisher Scientific, Waltham, MA, USA) were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred on to nitrocellulose membranes (Bio‐Rad). Membranes were probed with antibodies (Supplementary table 2 ) and then developed by chemiluminescence using Clarity ECL (Bio‐Rad).
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