Cells were rinsed with PBS and lysed with Laemmli sample buffer (50 mM Tris pH 6.8, 2% SDS, 0.025% Bromophenol Blue, 10% glycerol, 5% BME) and boiled for 10 min before separation by 10%–15% SDS–PAGE and transfer to polyvinylidene difluoride membranes (#162-0177 from Bio-Rad). Blots were then blocked in 5% milk for 1 h, probed with primary antibodies and horseradish peroxidase (HRP) conjugated secondary antibodies, and developed with SuperSignalTM West Dura Substrate (#34075 from Thermo Fisher Scientific).
Supersignal west dura substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
SuperSignal West Dura substrate is a chemiluminescent detection reagent used for Western blotting applications. It provides a stable, high-intensity signal that can be detected using common imaging systems. The product is designed to deliver consistent and reliable performance for the identification and quantification of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (5 mentions)
Las 3000 life science imager, by Fujifilm (4 mentions)
Protran, by Cytiva (4 mentions)
Nitrocellulose membrane, by Bio-Rad (4 mentions)
Trans blot turbo pvdf nitrocellulose membrane, by Bio-Rad (4 mentions)
Tgx gradient gel, by Bio-Rad (4 mentions)
Iblot2 system, by Thermo Fisher Scientific (4 mentions)
Gapdh, by Abcam (4 mentions)
Anti vinculin, by Merck Group (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
C myc, by Santa Cruz Biotechnology (3 mentions)
Laemmli buffer, by Bio-Rad (3 mentions)
Fusion solo s, by Vilber (3 mentions)
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
β actin, by Cell Signaling Technology (3 mentions)
Las 4000, by Cytiva (3 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (3 mentions)
β actin, by Merck Group (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
β actin, by Santa Cruz Biotechnology (3 mentions)
107 protocols using supersignal west dura substrate
1
Western Blot Protein Detection
2
Western Blot Analysis of EGFR and HIF1α
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Protein samples were resolved on NuPAGE Novex 4–12% Bis-Tris Gradient Gel (Invitrogen, Waltham, MA, USA) by SDS gel electrophoresis and transferred to polyvinylidene difluoride membrane (PVDF). Primary antibodies used in this study were as follows: 1:1000 dilution of EGFR (2232) and phospho-EGFR (Tyr 1173) (4407) antibodies from Cell Signalling Technology (Danvers, MA, USA), 1:500 dilution of HIF1α antibody from BD Bioscience (Bedford, MA, USA; 610959), 1:10,000 dilution of β-actin antibody from Sigma-Aldrich (A5441). Horseradish peroxidase-conjugated goat anti-mouse (170–6516) and goat anti-rabbit (170–6515) secondary antibodies were purchased from Bio-Rad (Hercules, CA, USA) and used at a 1:10,000 dilution. Primary antibodies were incubated with PVDF membranes overnight at 4 °C (with the exception of β-actin antibody - 1 h at room temperature). Secondary antibodies were incubated for 1 h at room temperature. Protein bands were detected with chemiluminescence using SuperSignalTM West Dura Substrate (34075, Thermo Fisher Scientific, Waltham, MA, USA) and visualised using a Bio-Rad VersaDoc Imaging System or a Bio-Rad ChemiDoc Imaging System. The intensity of bands was quantified with Image Lab V 5.2.1 software (Bio-Rad) and normalised to β-actin controls. Phosphorylated proteins were then normalised to their respective total protein level.
3
Western Blot Protein Analysis
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Cells were rinsed with PBS and lysed with Laemmli sample buffer (50 mM Tris pH 6.8, 2% SDS, 0.025% Bromophenol Blue, 10% glycerol, 5% BME) and boiled for 5–10 min before separation by 10–15% SDS–PAGE and transfer to polyvinylidene difluoride membranes (#162‐0177 from Bio‐Rad). Blots were then blocked in 5% milk for 1 h, probed with primary antibodies and horseradish peroxidase (HRP) conjugated secondary antibodies, and developed with SuperSignalTM West Dura Substrate (#34075 from Thermo Fisher Scientific).
4
SDS-PAGE and Immunoblotting Protocol
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Cells were rinsed with PBS and lysed using the Laemmli sample buffer (50 mM Tris pH 6.8, 2% SDS, 0.025% Bromophenol Blue, 10% glycerol, 5% BME). Lysates were boiled for 20 min, resolved using NuPAGE 4–12% SDS–PAGE gels (Life Technologies) and transferred to PVDF membranes (Millipore). Membranes were blocked using the Superblock T20 (TBS) Blocking Buffer (#37536 from Thermo Fisher Scientific), probed with primary antibodies overnight at 4 °C, and horseradish peroxidase (HRP) conjugated secondary antibodies at room temperature for 1 h. The immune complexes were detected by SuperSignalTM West Dura Substrate (#34075 from Thermo Fisher Scientific).
5
Dvl2, LRP6 Immunoblot Analysis
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Cells were lysed using 50 mM Tris-HCl, 150 mM NaCl, 1% sodium deoxycholate, 0.25 mM EDTA (pH 8.0), 1% Triton X-100, 0.2% sodium fluoride and protease inhibitor cocktail (Sigma, St Louis, MO, USA). Dvl2 (cat# 3224 S), pLRP6 (cat# 2568 S), LRP6 (cat# 3395 S), anti-rabbit IgG-HRP (cat# P0448), anti-mouse IgG-HRP (cat# P0447) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Immunoblots on PVDF were developed using SuperSignal West Dura substrate (Thermo Scientific, Rockford, IL, USA). The images were captured using the LAS-3000 Life Science Imager (Fujifilm; Tokyo, Japan).
6
Preparation and Analysis of Cell Lysates
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Cell lysates were prepared from exponentially growing cultures (OD600 = 0.6–0.8) as previously described (Wan et al., 2013 (link)) with the following modifications. The equivalent of 1.0 ml of culture at an OD600 of 0.6–0.8 was centrifuged at 16,000 × g for 5 min at 4°C. The supernatant was removed, and cell pellets were resuspended in 50 μl of 10mM Tris pH 8.0, followed by the addition of 50 μl of 2x SDS sample buffer. Samples were boiled for 5 min at 100°C before being run on a 12% (w/v) polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were blocked for 30 min in 5% (w/v) non-fat dry milk in TBST (20 mM Tris, pH 8, 0.05% (w/v) Tween 20), and incubated at 4°C overnight with primary antibodies. Anti-FLAG tag and McpA antibodies were used at a concentration of 1:10,000. Then, a 1:10,000 dilution of secondary antibody, HRP-conjugated goat anti-rabbit immunoglobulin, was incubated with the membranes at room temperature for 2 h. Membranes were developed with SuperSignal West Dura Substrate (Thermo Scientific, Rockford, IL).
7
Western Blot Analysis of HA-Treated Cells
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Whole‐cell extracts from HA‐treated HPF and HGF cells were prepared by lysis in RIPA buffer as described.30 Lysates were run on 10% SDS‐PAGE, and transferred to Amersham™ Protran® membrane (Sigma, Basel, Switzerland). Proteins of interest were visualized using anti‐phospho‐Akt, anti‐Akt, anti‐phospho‐Erk1/2, anti‐Erk, anti‐phospho‐p38, anti‐p38 (all from Cell Signaling Technology, Danvers, MA, USA), and anti‐vinculin (Sigma) antibodies followed by horseradish peroxidase‐conjugated secondary antibodies (MP Biomedicals, Santa Ana, CA, USA) for detection with the SuperSignal™ West Dura Substrate (ThermoFisher Scientific, Zug, Switzerland). Phospho‐Akt, phospho‐Erk1/2 or phospho‐p38 protein expression relative to the respective total protein control was quantified by densitometry using ImageQuant (Molecular Dynamics, Groningen, The Netherlands). Data represent means ± SD from three independent experiments performed with three different cell donors.
8
Western Blotting Protein Analysis Protocol
9
Western Blot Analysis of THP-1 Cell Proteins
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THP-1 cells were treated with SEB and lysed, and their total protein content was determined as described in Caspase Activity Assay Section. Approximately 25 μg of protein was separated through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidine difluoride (PVDF) membrane (Mini Trans-Blot Cell, BioRad). The PVDF membrane was blocked with 5% (m/v) non-fat milk (Boster, Wuhan, China) in TBST buffer (50 mM Tris, 138 mM NaCl, 2.7 mM KCl, pH 8.0, with 0.05% (v/v) Tween 20; ZSGB-BIO, Beijing, China) for 1 h and incubated in anti-HLA-DRa (1:200; Santa Cruz Biotechnology, Paso Robles, CA, USA), anti-TNFR1 (1:200; Santa Cruz), and anti-β-actin (1:1000, Boster) diluted in 5% (m/v) non-fat milk at 4°C overnight. The membrane was washed five times with TBST, incubated in goat-anti-mouse secondary antibody conjugated with horseradish peroxidase (HRP; 1:3000, Boster) diluted in 5% (m/v) non-fat milk for 1 h, and washed five times with TBST. Immunoreactivity was visualized by using a SuperSignal West Dura substrate (Thermo Fisher Scientific). Bands were detected by using ChemiDoc Touch Imaging System (BioRad) and densitometrically analyzed with ImageJ 1.51c (Wayne Rasband, National Institutes of Health, USA).
10
Western Blot Analysis of Tsetse Fly Saliva
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~2 μg of G. m. morsitans salivary proteins were treated with PNGase F (New England Biolabs), resolved by SDS-PAGE and transferred onto a PVDF membrane (GE Healthcare) at 90V for 1 hour. After verification of transfer with Ponceau Red (Sigma-Aldrich), the membrane was blocked for 1 hour (PBS-T [Sigma, US] containing 5% skim milk powder [Sigma]) and incubated in 1:10,000 rabbit-anti-G. m. morsitans saliva antibody overnight at 4°C. Membranes were washed and probed at room temperature for 1 hour with 1:20,000 HRP-labelled goat-anti-rabbit antibody (ThermoFisher). Super Signal West Dura substrate (ThermoFisher) was used for detection.
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