Primary MEFs were lysed in RIPA buffer (CST, catalog no. 9806S) supplemented with a protease inhibitor (Thermo Fisher, catalog no. 78430) and phosphatase inhibitor (Thermo Fisher, catalog no. 88667). An equal amount of protein was loaded and separated on 10% SDS-PAGE gels (Bio-Rad), then transferred to polyvinylidene fluoride membranes (EMD Millipore). Primary antibody against RelB (clone C1E4), IκBα (clone L34A5), and GAPDH were stained and detected by the use of horseradish-peroxidase-conjugated secondary anti-rabbit antibody (Invitrogen, catalog no. 31460). All the blots were performed using PierceTM ECL Substrate (Thermo Fisher Scientific) and scanned with a ChemiDocTM Touch Imaging System (Bio-Rad).
Pierce ecl substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
The Pierce ECL substrate is a chemiluminescent detection reagent used for the visualization of proteins in Western blotting applications. It generates a luminescent signal when exposed to the horseradish peroxidase (HRP) enzyme, which is commonly used to label primary or secondary antibodies in immunodetection procedures.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Laemmli sample buffer, by Bio-Rad (5 mentions)
Chemidoc touch imaging system, by Bio-Rad (4 mentions)
Pvdf membrane, by Bio-Rad (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Cl xposure film, by Thermo Fisher Scientific (3 mentions)
Water bath sonicator, by Diagenode (3 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (3 mentions)
Nupage mops running buffer, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail set 3, by Merck Group (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Chemidoc imaging system, by Bio-Rad (3 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Hrp conjugated goat anti mouse igg, by Proteintech (2 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Hrp conjugated goat anti rabbit igg, by Proteintech (2 mentions)
Iblot 2 gel transfer device, by Thermo Fisher Scientific (2 mentions)
Bis tris gradient gel, by Thermo Fisher Scientific (2 mentions)
Novex semi dry apparatus, by Thermo Fisher Scientific (2 mentions)
103 protocols using pierce ecl substrate
1
Western Blot Analysis of Signaling Proteins
2
Quantitative Western Blot Analysis
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The western blot analysis was conducted according to a previously reported
method19 (link). The concentration of
extracted proteins was measured by BCA Protein Quantification Kit (wellbio).
After denaturation in boiling water, protein samples (30 μg) were loaded onto
polyacrylamide gel and separated. Following electrophoresis, the proteins were
transferred onto polyvinylidene difluoride (PVDF) membrane, and then the
membranes were blocked with Tris-buffered saline Tween-20(TBST) containing 5%
skimmed milk powder at room temperature for 1.5 h. Subsequently, the membranes
were incubated with each primary antibody against IL-1β (1:1000), IL-6 (1:1000),
TNF-α (1:1000), TLR4 (1:500), NF-κB (1:50000), Bax (1:2000), casapase-3
(1:1000), casapase-9 (1:500), ZO-1 (1:500), occludin (1:500) and Claudin-1
(1:2000) overnight at 4 °C. After three washings with TBST for 15 min each, the
membranes were incubated with the appropriate horseradish peroxidase (HRP)
conjugated goat anti-rabbit IgG (1:6000, Proteintech Group) or HRP conjugated
goat anti-mouse IgG (1:5000, Proteintech Group) for 1 h at room temperature.
Immunoreactivity was reacted with a PierceTM ECL substrate (thermo) for 3 min
and then exposed to X-ray films. The results were standardized to the intensity
levels of β-actin and analyzed using the Quantity-One software (Bio-Rad,
Hercules, CA, United States).
method19 (link). The concentration of
extracted proteins was measured by BCA Protein Quantification Kit (wellbio).
After denaturation in boiling water, protein samples (30 μg) were loaded onto
polyacrylamide gel and separated. Following electrophoresis, the proteins were
transferred onto polyvinylidene difluoride (PVDF) membrane, and then the
membranes were blocked with Tris-buffered saline Tween-20(TBST) containing 5%
skimmed milk powder at room temperature for 1.5 h. Subsequently, the membranes
were incubated with each primary antibody against IL-1β (1:1000), IL-6 (1:1000),
TNF-α (1:1000), TLR4 (1:500), NF-κB (1:50000), Bax (1:2000), casapase-3
(1:1000), casapase-9 (1:500), ZO-1 (1:500), occludin (1:500) and Claudin-1
(1:2000) overnight at 4 °C. After three washings with TBST for 15 min each, the
membranes were incubated with the appropriate horseradish peroxidase (HRP)
conjugated goat anti-rabbit IgG (1:6000, Proteintech Group) or HRP conjugated
goat anti-mouse IgG (1:5000, Proteintech Group) for 1 h at room temperature.
Immunoreactivity was reacted with a PierceTM ECL substrate (thermo) for 3 min
and then exposed to X-ray films. The results were standardized to the intensity
levels of β-actin and analyzed using the Quantity-One software (Bio-Rad,
Hercules, CA, United States).
3
Bile Acid-Induced ER Stress Signaling
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HET1A cells were treated with bile acids for 6 h. For PP2 pre-treatment studies PP2 (10 uM) was added 24 h prior to co-treatment with bile acids for 6 h. For salubrinal and GSK2606414 pre-treatment studies, these compounds were added 1 h prior to co-treatment with bile acids. Cells were lysed with NP40 buffer and protein quantified using a PierceTM BCA kit (Thermo Fisher Scientific Waltham, MA USA). Equal concentrations of protein were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% Marvel, primary antibodies; pPERK/Total PERK/total IRE-1/pSrc/total src/pEFI2a/total EIF2α/tubulin/actin (Cell signalling Technologies Inc. Danvers, MA, USA) or pIRE-1 (Abcam, Cambridge, United Kingdom) were incubated overnight in 5% BSA/TBST. HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Inc. Dallas, Texas) were incubated for 1 h and detected using PierceTM ECL substrate (Thermo Fisher Scientific, Waltham, MA USA).
4
Western Blot Analysis of Hippocampus
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Mice (part 1: at post-ischemic 24 h) or rats (part 2: at post-ischemic day 28) were decapitated under anesthesia, the hippocampus was separated from the lesion hemisphere, frozen quickly in liquid nitrogen, and stored at -80°C. The hippocampus tissues were disrupted in PBS and homogenized with RIPA buffer by ice-cold Immune Precipitation Assay and then centrifuged at 12000 × g for 15 min. Protein samples of 50 μg per lane were separated on polyacrylamide gel and then transferred onto a polyvinylidene difluoride (PVDF) membrane. Membranes were blocked with a 5% milk solution (non-fat dry milk in TBST) for 1 h and then incubated with specific antibodies as described above at 4°C overnight. Following five washes with TBST for 15 min each, the membranes were incubated with the corresponding HRP conjugated goat anti-mouse IgG (1:4000, Proteintech Group) or HRP conjugated goat anti-rabbit IgG (1:6000, Proteintech Group) for 1 h at room temperature. Immunoreactivity was detected with a PierceTM ECL substrate (Thermo) for 3 min, and then, the samples were exposed to X-ray films. The values were normalized to the intensity levels of β-actin and analyzed using Quantity-One software (Bio-Rad, Hercules, CA, United States).
5
Western Blot Analysis of FMDV Proteins
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FMDV samples were mixed with 4× Lithium dodecyl sulfate (LDS) sample buffer (Invitrogen, Carlsbad, CA, USA). The pretreated samples were heated at 70 °C for 10 min. Samples were run on 4–12% gradient bis–tris gels (Invitrogen) transferred to a nitrocellulose membrane using the iBlot2 gel-transfer device (Invitrogen). The membranes were blocked in phosphate-buffered saline with Tween-20 (PBST, 10 mM sodium phosphate, 132 mM NaCl, 2.7 mM KCl, and 0.05% Tween-20, pH 7.4). The membranes were washed and incubated with anti-FMDV type O VP1 (76.5E) and anti-FMDV 3B (4G24) monoclonal antibodies, produced in-house, diluted 1/2000 in PBST, followed by incubation with goat anti-mouse horseradish peroxidase-conjugated secondary antibodies (Invitrogen) diluted 1/4000 in PBST. Proteins were visualized with Pierce ECL Substrate (Invitrogen) using an Azure C600 imaging system and the cSeries Capture Software (Azure Biosystem, Dublin, CA, USA).
6
Western Blot Analysis of FMDV Proteins
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The protein samples were mixed with 4× lithium dodecyl sulfate sample buffer (Invitrogen, Carlsbad, CA, USA) and heated at 90 °C for 10 min. Samples were run on 4–12% gradient bis–tris gels (Invitrogen) and then transferred to a nitrocellulose membrane using the iBlot2 gel-transfer device (Invitrogen). The membranes were blocked in buffer [PBST; 10 mM sodium phosphate, 132 mM NaCl, 2.7 mM KCl, and 0.05% Tween-20, pH 7.4] for 1 h at 25 °C with shaking, washed three times with PBS-T for 10 min, and then incubated with a home-made primary antibody against FMDV VP1 (76.5E) and 3B (4G24) diluted 1/2000 in PBST at 4 °C overnight. The membranes were washed three times with PBS-T and incubated with HRP-conjugated goat anti-mouse secondary antibody (Invitrogen) diluted 1/4000 in PBST for 1 h at RT. Proteins were detected with Pierce ECL Substrate (Invitrogen) using an Azure C600 imaging system and cSeries Capture Software (Azure Biosystems, Dublin, CA, USA).
7
Quantitative Analysis of KCa3.1 Protein
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KCa3.1 protein expression in primary HBECs was analysed by Western blotting as described previously [28 (link)]. Soluble protein from equivalent numbers of cells was resolved by 10% SDS-PAGE and then transferred to a nitrocellulose membrane. Membranes were blocked and assayed by immunoblot using 1 μg/ml anti-KCa3.1 antibody (Sigma). Protein bands were identified by goat anti-rabbit IgG horseradish peroxidase (HRP)-linked secondary antibody (Dako, Ely, UK). Bands were visualised by chemiluminescence with Pierce ECL substrate (Invitrogen) and the ImageQuant LA S 4000 (GE Healthcare Life Sciences, Little Chalfont, UK). Β-actin was used as a positive control for the western blot. To re-stain blots, bound primary and secondary antibodies were removed by incubating the membrane in 0.2 M NaOH for 5 min. Membranes were washed, blocked, probed with 6.7 ng/ml primary monoclonal β-actin antibody HRP conjugate (Santa Cruz Biotechnology, Dallas, Texas, USA) for 2 h at room temperature, and then visualised by chemiluminescence as described above.
8
Western Blot Analysis of GLI2 Protein
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The resulting IP eluates and total protein (input) samples were separated using a 4–12 % denaturing polyacrylamide gel (NuPAGE, Invitrogen) with MOPS running buffer according to the manufacturer’s protocol. The resolved proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad), blocked using non-fat dry milk (Carnation) reconstituted with Tris-buffered saline and Tween 20 (TBST), and incubated with GLI2- (kind gift from C.C. Hui [47 (link)]), Brg1- (Millipore), FLAG- (Sigma-Aldrich), or α-tubulin-specific (DM1A, Sigma-Aldrich) antibodies. Membranes were stripped with re-blot plus mild stripping buffer (Millipore) between each primary antibody incubation. The signal was detected using horseradish peroxidase (HRP)-conjugated secondary anti-mouse (Cell Signalling) or anti-rabbit (Santa Cruz) antibodies, followed by a chemiluminescence reaction using Pierce ECL substrate (Fisher Scientific).
Densitometry was performed on the GLI2-specific bands with the ImageJ program (National Institutes of Health, USA) [48 ]. The densities of the GLI2 protein in the FLAG-IP samples were normalized to the loading control, α-tubulin, and were presented as a percentage of the highest band density.
Densitometry was performed on the GLI2-specific bands with the ImageJ program (National Institutes of Health, USA) [48 ]. The densities of the GLI2 protein in the FLAG-IP samples were normalized to the loading control, α-tubulin, and were presented as a percentage of the highest band density.
9
Western Blot Analysis of LYPLAL1 and HSP90
10
Western Blot Analysis of LYPLAL1 and HSP90
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Proteins separated by SDS-PAGE were transferred onto membranes using a Novex semi-dry apparatus (Life Technologies) and membranes blocked in 5% milk-TBST. Blots were incubated overnight at 4°C with primary antibodies (LYPLAL1 1:1000 dilution, Proteintech 16146-AP; HSP90 1:2000 dilution, Genetex GTX109753) diluted in 5% BSA-TBST. After washing and incubation with HRP-conjugated secondary antibodies, blots were treated with Pierce ECL substrate (Fisher Scientific) and developed.
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