In-Cell Western Assay (Li-Cor, Lincoln, Nebraska) was performed according to the manufacturer’s instructions and as previously described.9 (link) Cells were incubated with antibodies against p-IκBα (1/1000) (Cell Signaling Technology, Danvers MA) and β-actin (1/1000) (Santa Cruz Biotechnology, Birmingham AL) as a loading control for 2 hours. This was followed by incubation with secondary antibodies IRDye800CW donkey anti-goat (1/1000) and IRDye680RD donkey anti-rabbit (1/1000) (Li-Cor) for 1 hour with protection from light. Plates were scanned, florescence detected at 700 and 800 nm, and data normalized using an Odyssey CLx Infared Imaging System.
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In-Cell Western Assay for Protein Analysis
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Corresponding Organization : Child Health and Development Institute
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Variable analysis
independent variables
- Antibodies against p-IκBα and β-actin
- Fluorescence detected at 700 and 800 nm
- Incubation time (2 hours for primary antibodies, 1 hour for secondary antibodies)
- Antibody concentrations (1/1000 dilution)
- Scanning and data normalization using Odyssey CLx Infrared Imaging System
- β-actin as a loading control
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