The left neocortex of each animal was collected for protein extraction after saline perfusion and disection. Tissue was homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors (Roche Diagnostic). The protein concentration of the cortical lysate was measured and equal amounts (20 – 50 μg per lane) were loaded onto 10 – 15% SDS-polyacrylamide gels for electrophoresis. Proteins were transferred onto PVDF membranes (Bio-rad) for antibody staining. Membranes were incubated with primary antibodies after blocking with non-fat milk (Table S3 ), and probed with the appropriate horseradish peroxidase-linked secondary immunoglobulin (Cell signalling). GAPDH served as a loading control. The immunoblots were visualized by chemiluminescent substrates (SuperSignal™ West Pico or West Femto Substrates, Thermo Scientific) and signals were detected using medical blue sensitive X-ray films. The films were digitalized and the protein intensities were quantified using Image J software.
West femto substrates
Manufactured by Thermo Fisher Scientific
The West Femto Substrates are laboratory consumables designed for use in western blotting applications. They serve as the substrate for chemiluminescent detection of proteins on western blot membranes. The substrates provide a sensitive and stable luminescent signal to enable visualization of target proteins.
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Lab products found in correlation
2 protocols using west femto substrates
1
Western Blot Analysis of Cortical Proteins
2
Western Blot Protein Analysis Protocol
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The left neocortex of each mouse or a human tissue section removed from a slide was lysed for protein extraction using RIPA buffer (radioimmunoprecipitation assay buffer) supplemented with phosphatase and protease inhibitors (Roche Diagnostic). Protein sample concentrations were quantified by bicinchoninic acid assay (Bio-Rad) and normalized amounts of proteins (20 -50 μg) were electrophoresed on a 10 -15% SDS-polyacrylamide gels. After transfer to PVDF membranes, non-specific binding was blocked with 5% non-fat milk followed by primary antibody incubation (Table S3). The membranes were probed with horseradish peroxidaselinked secondary immunoglobulins (Cell signalling) before visualization with chemiluminescent substrates (SuperSignal™ West Pico, Dura or West Femto Substrates, Thermo Scientific). The chemiluminescent signals were detected by blue sensitive medical Xray film, and the resulting band densities were digitalized for quantification using ImageJ software. GAPDH was used as the protein loading control. For immunoprecipitation, proteins of interests were pulled down by Dynabeads™ Protein G (ThermoFisher) using specific antibody prior to western blotting with target antibody.
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