Freshly isolated neutrophils were stimulated with GM-CSF (50 ng/mL) for the times indicated in the figure legends, and the cells were washed by ice-cold phosphate-buffered saline and lysed with RIPA Buffer (Sigma-Aldrich) supplemented with 1.0 mM sodium orthovanadate, 10 μg/mL aprotinin, and 10 μg/mL leupeptin for 20 min at 4 °C. After 5 min on ice, the cell lysates were centrifuged at 10,000g for 10 min at 4 °C. After centrifugation, cellular lysates (30 μg) were also subjected to 12% SDS-PAGE followed by Western blot with antibodies against human NLRP3 or β-actin with an ECL Western blotting kit (Amersham, Little Chalfont, UK). Only in the signal transduction analysis, cells were pretreated with tofacitinib for 30 min and then stimulated with GM-CSF. Western blot analysis using phospho-specific anti-JAK and STAT antibodies was performed with an ECL Western blotting kit (Amersham).
Ecl western blotting kit
Manufactured by Cytiva
Sourced in United Kingdom, United States
The ECL Western blotting kit is a laboratory equipment used for the detection and analysis of proteins in Western blot experiments. It provides the necessary reagents and solutions for the chemiluminescent detection of target proteins on a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (9 mentions)
Hybond, by Cytiva (9 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Gapdh, by Santa Cruz Biotechnology (6 mentions)
Immobilon p membrane, by Merck Group (5 mentions)
Polyvinylidene difluoride membrane, by Pall Corporation (4 mentions)
Hybond ecl nitrocellulose membrane, by Cytiva (4 mentions)
Anti actin, by Santa Cruz Biotechnology (4 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Protease phosphatase inhibitor cocktail, by Abcam (3 mentions)
Amersham hybond ecl nitrocellulose membranes, by Cytiva (3 mentions)
Ripa buffer, by Merck Group (2 mentions)
Aprotinin, by Merck Group (2 mentions)
Chemidoc mp system, by Bio-Rad (2 mentions)
Hrp conjugated anti rabbit igg, by Santa Cruz Biotechnology (2 mentions)
Goat anti rabbit igg hrp, by Abmart (2 mentions)
Anti tubulin, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh rabbit polyclonal antibody, by Santa Cruz Biotechnology (2 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Gel logic 1500 imaging system, by Kodak (2 mentions)
55 protocols using ecl western blotting kit
1
Neutrophil NLRP3 Activation by GM-CSF
2
Protein Expression Analysis of Lung Tissue
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The frozen right lung tissue were lysed using protein extraction buffer and equal amounts of protein were denatured and separated by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis (SDS-PAGE). Protein concentrations were assessed using the BCA Protein Assay kit (Santa Cruz Biotechnology). 10μg of total protein were electrophoresed on 4-20% gradient SDS-PAGE gels and transferred to a nitrocellulose membrane. The membrane was blocked for 16 h at 4°C in blocking buffer containing 5% skim milk powder in TBST [20 mM Tris HCl (pH 7.4). Blots were probed with specific primary antibodies TGF-β1(ab25121), Smad2(ab63576), Smad3 (ab40854), and p-Smad2(ab188334), p-Smad3(ab52903), VEGF (ab46154), Smad7 (sc-11392; Santa Cruz Biotechnology, Inc), α-SMA (ab5694), E-cadherin (sc-7870), N-cadherin (sc-7939), TIMP-1 (ab61224). The goat anti-rabbit IgG (Boshide Inc., Shanghai, China) were incubated at 37°C for 1 h as the secondary antibody. Immunoreactions were visualized using an chemiluminescence (ECL) Western blotting kit (Amersham Biosciences) following the manufacturer′s recommendations. (Thermo Fisher Scientific Co., Ltd., Shanghai, China).
3
Immunoblotting Analysis of CYB5D2 Protein
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Cells lysates were produced in a lysis buffer consisting of 20 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EGTA, 1 mM EDTA, 25 mM sodium pyrophosphate, 0.1 mM sodium orthovanadate, 1 mM β-glycerophosphate, 1 mM NaF, 2 μg/ml leupeptin, 1 mM PMSF and 10 μg/ml aprotinin (Sigma Aldrich, Oakville, ON). Total cell lysate protein (50 μg) was separated on an SDS-PAGE gel and transferred onto nitrocellulose membranes (Amersham, Baie d’Urfe, QC), which were blocked with skim milk (5%) followed by incubation with antibodies to CYB5D2 (1:1000) or actin (Santa Cruz, 1:1000) at 4 °C overnight. Signal was developed using HRP-conjugated secondary antibodies and an ECL Western Blotting Kit (Amersham, Baie d’Urfe, QC)30 (link). We quantified protein bands with ImageJ (National Institutes of Health).
4
Protein Analysis via Western Blot
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Total protein from cells was extracted using RIPA buffer (Beyotime). An equal amount of each protein sample was separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 5% nonfat milk at room temperature for 1 h and incubated with specific primary antibody, ALDOA (1:2000, Abcam, Cambridge, UK) and GAPDH (1:5000, Santa Cruz, CA, USA) overnight, followed by incubation with HRP-conjugated secondary antibodies (Santa Cruz, CA, USA). The protein bands were detected using ECL western blotting kit (Amersham Biosciences, Buckinghamshire, UK). GAPDH was used as the loading control.
5
Western Blot Analysis of PI3K/Akt/mTOR Pathway
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Western blot analysis was cultured as previously described [16 (link)]. GALNT7 monoclonal antibody (1:1000, Abcam, Cambridge, UK), PI3K, p-PI3K Tyr458, Akt, p-Akt Ser473, mTOR, p-mTOR Ser2248 antibody (1:1000 Proteintech, Chicago, USA). Immunoreactive bands were visualized using ECL Western blotting kit (Amersham Biosciences, Buckinghamshire, UK) and were normalized to GAPDH.
6
Protein Expression Analysis by Western Blot
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The entire protein lysates were split by 10% SDS‐PAGE. The split protein was transferred into a PVDF membrane (Millipore, Bedford, MA, USA). The above membranes were incubated with the primary Abs SMAD2 (1:1000 dilution; Abcam, Cambridge, UK) and GAPDH (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight. The membranes were then treated with secondary Ab conjugated to HRP for 2 hours at room temperature. Detection was undertaken using an ECL western blotting kit (Amersham Biosciences, Little Chalfont, UK). GAPDH was used as the loading control. The intensities of protein bands were quantitated using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
7
Histone Acetylation Modulation in HEK293 Cells
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HEK 293 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (PAA Laboratories GmbH, Pasching, Austria). The cultures were incubated at 37°C in a humidified atmosphere of 5% CO2. HEK293 cells were seeded on 32-mm round glass coverslip at a density of 2 × 105 cells. At ∼70% of confluence, cells were transfected with a total amount of 1 μg of expression vectors using Nanofectin I (PAA Laboratories, Inc). Twenty-four hours later, coverslips were mounted in an open observation chamber with DMEM-F12 without Phenol red, B12 vitamin and Riboflavin, and supplemented with 20 mM HEPES and l -glutamine (PAA). The histone deacetylase inhibitor sodium butyrate (NaB), sodium azide (Az), and deoxy-glucose (DG) were obtained from Sigma Aldrich (Stenheim, Germany). For NaB treatment, cells were incubated 24 h after transfection with 2.5 mM of NaB for 24 h before imaging. For ATP depletion, cells were incubated with 10 mM Az and 10 mM DG for 2 h before imaging. Histone acetylation was verified after acid–urea–Triton polyacrylamide gel electrophoresis (15% acrylamide, 0.1% M-bisacrylamide, 6 M urea, 5% acetic acid, 8 mM Triton X-100, 0.5% TEMED, 0.00025% riboflavin) by western blot with an anti-H4 primary antibody (07-108, Upstate, France), and peroxidase-conjugated secondary antibody (Amersham ECL western blotting kit) (Supplementary Figure S1A ).
8
Neutrophil Inflammatory Response Assay
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Neutrophils (1×106) were seeded in 24-well plates containing RPMI1640 supplemented with 10% heat-inactivated FBS and stimulated with SAA for 8 hours. Cell-free supernatants were collected by centrifugation at 400 g for 5 minutes. The supernatants or cellular lysates (50 µg) were also subjected to 12% SDS-PAGE, followed by western blot with antibodies against human IL-1β or caspase-1 with an ECL Western blotting kit (Amersham, Little Chalfont, UK).
9
Western Blot Analysis of Cell Signaling
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A total of 30 μg of each cell or tissue lysate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were subsequently transferred to Hybond membranes (Amersham Pharmacia Biotech, Buckinghamshire, UK). The membranes were blocked and then incubated with the designated primary antibodies, and signals were detected using an ECL Western Blotting Kit (Amersham Pharmacia Biotech). Goat anti-BAMBI polyclonal antibody (R&D Systems, Minneapolis, MN, USA), anti-TGF-β1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) anti-β-catenin (BD Biosciences, Franklin Lakes, NJ, USA), anti-HBx (Chemicon International, Temecula, CA, USA) and β-actin (Sigma, Saint Louis, MO, USA) were used as the primary antibodies.
10
Western Blot Analysis of EMT Markers
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Cells were lysed using ice-cold radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China), and the protein concentration was measured using a BCA Protein Assay Kit (Solarbio, Beijing, China). Equal amounts of protein were fractionated by SDS-PAGE gel and then transferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, U.S.A.). After blocking with 5% skim milk, the membranes were incubated overnight at 4°C with specific primary antibodies against SOX4 (1:1000; Santa Cruz Biotechnology, Inc., Dallas, TX, U.S.A.), E-cadherin (1:1000; Abcam, Cambridge, U.K.), N-cadherin (1:1000; Abcam), Vimentin (1:1000; Abcam), and GAPDH (1:1000; Santa Cruz Biotechnology, Inc.). After rinsing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The signals were visualized using an ECL western blotting kit (Amersham Biosciences, Piscataway, NJ, U.S.A.), and GAPDH was set as the internal control.
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