Cell lysates, serial dilutions of standards, and positive and negative controls were arrayed on nitrocellulose-coated slides (Grace Bio-Labs) using the Quanteriz 2470 Arrayer. Each slide was probed with a validated primary antibody plus a biotin-conjugated secondary antibody. Signal detection was amplified using an Agilent GenPoint staining platform and visualized by DAP colorimetric reaction. The slides were scanned, analyzed, and quantified to generate spot intensity using customized software (Array-Pro Analyzer, Media Cybernetics). Each dilution curve was fitted with a logistic model (RPPA SPACE developed at MD Anderson). The protein concentrations were then normalized for protein loading. The corrections factor was calculated and normalized across sets via replicates-based normalization using an invariant set of control samples to adjust for batch differences between identical controls.26 (link)
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Quantitative Protein Profiling Using RPPA
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Other organizations : Baylor College of Medicine, Texas Children's Hospital
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Variable analysis
independent variables
- Cell lysates
- Serial dilutions of standards
- Spot intensity
- Positive controls
- Negative controls
- Positive controls
- Negative controls
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