Ba/F3 cells expressing various EGFR mutants were treated with varying doses of inhibitors for 8 hours. Cells were then harvested and lysed in RIPA buffer. Protein quantification was performed using Pierce BCA protein assay (Thermo Fisher). 10 μg protein lysate was resolved on NuPAGE 4-12% Bis-Tris gels (Thermo Fisher), followed by transfer onto PVDF membranes (Millipore). Membranes were blocked with 3% BSA for 30 minutes on a rocking platform at room temperature. Membranes were subsequently incubated overnight at 4°C with primary antibodies for phospho-EGFR (Cell Signaling #3777), total EGFR (Cell Signaling #4267), phospho-AKT (Cell Signaling #4060), total AKT (Cell Signaling #9272), phospho-ERK1/2 (Cell Signaling #4377), total ERK1/2 (Cell Signaling #9102), and HSP90 (Cell Signaling #4877). The following morning, membranes were incubated with anti-rabbit secondary antibody (Cell Signaling #7074) and imaged on an Amersham Imager 600 chemiluminescence imager using SuperSignal West Dura ECL substrate (Thermo Fisher).
Supersignal west dura ecl substrate
Manufactured by Thermo Fisher Scientific
SuperSignal West Dura ECL substrate is a chemiluminescent detection reagent designed for Western blot applications. It provides a sensitive and long-lasting signal for the detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Sds page, by Thermo Fisher Scientific (2 mentions)
A6154, by Merck Group (2 mentions)
S9684, by Merck Group (2 mentions)
Genesys image acquisition software, by Syngene (2 mentions)
Anti rabbit secondary antibody, by Cell Signaling Technology (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Total erk1 2, by Cell Signaling Technology (1 mentions)
Phospho egfr, by Cell Signaling Technology (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
Total egfr, by Cell Signaling Technology (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Hsp90, by Cell Signaling Technology (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Bistris nupage pre cast gels, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Intelligent dark box 2, by Fujifilm (1 mentions)
6 protocols using supersignal west dura ecl substrate
1
EGFR Mutant Cell Signaling Profiling
2
Protein Expression Analysis by Western Blot
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Cells and tissues were lysed in RIPA buffer (ThermoFisher Scientific) containing protease inhibitor cocktail (ThermoFisher Scientific). Samples were centrifuged and supernatant collected for protein analysis. After protein quantification with the BCA method, samples were separated using Bis-Tris NuPage precast gels (ThermoFisher Scientific) and transferred to PVDF membranes. After blocking with 5% nonfat milk for 1 hour, primary antibodies were incubated overnight at 4°C. Membranes were washed in TBS-tween buffer and incubated in HRP conjugated secondary antibodies for 1h at room temperature. Blots were developed using SuperSignal West Dura ECL substrate (ThermoFisher Scientific) and imaged using FluorChem M ProteinSimple imager.
3
Western Blotting Procedure for Protein Detection
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Equal volumes of cytoplasmic and nuclear extracts (adjusted by blotting with cytoplasmic and nuclear markers) were boiled in SDS sample buffer containing 10 mM DTT and separated by SDS-PAGE. Proteins were electrotransferred to polyvinylidene difluoride membrane (Immobilon P) and blocked for 1 h in 5% BSA in PBS containing 0.04% Tween 20. Membranes were incubated with primary antibody, diluted in 1% BSA in PBS at 4°C overnight, washed three times in PBS, and incubated with horseradish peroxidase–conjugated anti–rabbit (1:40,000 dilution) or anti–mouse (1:25,000 dilution) antibody (Amersham Pharmacia). Proteins were visualized using Super Signal West Dura ECL substrate (ThermoScientific), and membranes were scanned with a charge-coupled device camera Intelligent Dark Box II (Fujifilm). Raw data files were exported as TIFF files from Aida software, and level autocontrast was applied to the blots using Photoshop (Adobe).
4
SNAP25 Western Blot Analysis
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All samples were resolved by SDS-PAGE (Invitrogen, Carlsbad, CA) and transferred onto nitrocellulose membranes. Membranes were blocked with 5% nonfat milk in 0.1% PBS-T (phosphate buffered saline with Tween-20) and probed with an anti-SNAP25 antibody (1/2000; S9684, Sigma-Aldrich). Immunoreactive bands were detected using horseradish peroxidase–conjugated secondary anti-rabbit (1/2000; A6154, Sigma-Aldrich) antibodies and SuperSignal West Dura ECL substrate (Thermo Fisher Scientific). Bands were visualized on a Pxi4 imaging system using GeneSys image acquisition software (Syngene, Bangalore, Karnataka, India), and intensities of the total form and cleaved form of SNAP25 were measured with GeneTools (Philomath, OR, USA).
5
Western Blot Analysis of SNAP-25 Cleavage
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All samples were resolved by SDS-PAGE (Invitrogen, Carlsbad, CA, USA) and transferred onto nitrocellulose membranes. Membranes were blocked with 5% non-fat milk in 0.1% PBS-T (phosphate buffered saline with Tween-20) and probed with an anti-Firefly antibody (ab185924, Abcam, 1/5000) or anti-SNAP-25 antibody (S9684, Sigma-Aldrich, 1/2000). Immunoreactive bands were detected using horseradish peroxidase–conjugated secondary anti-rabbit (A6154, Sigma-Aldrich, 1/2000) antibodies and SuperSignal West Dura ECL substrate (Thermo Fisher Scientific). Bands were visualized on a Pxi4 imaging system using GeneSys image acquisition software (Syngene, Bangalore, Karnataka, India).
For SNAP-25 cleavage assay, intensities of the total form and cleaved form of SNAP-25 were measured with GeneTools (Philomath, OR, USA).
For SNAP-25 cleavage assay, intensities of the total form and cleaved form of SNAP-25 were measured with GeneTools (Philomath, OR, USA).
6
Rat E14 Spinal Cord Dissociation and Immunoblotting
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