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How RPPA works
RPPA Applications
RPPA Applications
- Cell or tissue lysates are arrayed on nitrocellulose-coated slides using Aushon 2470 Arrayer (Aushon BioSystems) and printed in triplicate (as technical replicate). Spotted slides are then blocked with an aerosol BSA solution and stored at 4°C in a desiccator. Immunolabeling is performed on an automated Slide Stainer. Each slide is incubated with a single primary antibody at room temperature for 30 min and followed by a secondary antibody. A negative control slide is incubated with antibody diluent instead of a primary antibody. A Signal Amplification System and fluorescent IRDye 680 Streptavidin (LI-COR) are used as the detection system. Total protein for each spot is assessed by staining one in every 20 slides with Sypro Ruby Blot Stain (Molecular Probes).
- The immunostained slides are imaged using a GenePix AL4200 scanner (at 635 nm wavelength for antibody slides or 535 nm wave length Sypro Ruby Blot-Stained slides), and the images are analyzed by GenePix Pro 7.2 software tools (Molecular Devices). The fluorescence signal of each spot is obtained after the local slide background signal is subtracted from the fluorescence intensity.
RPPA Applications
RPPA Applications
RPPA Applications
- Characterization of cellular signaling networks under various conditions
- Protein biomarker identification and validation in preclinical and clinical samples
- Drug target identification and validation
- Analysis of specificity of protein degradation by degrader compounds
- Mechanistic studies for various indications
- Determination of drug selectivity and identification of therapeutic targets
- Definition of regulatory mechanisms in signaling networks, including cross-talk and feedback loops
- Classification of patient tumors, prognosis determination, and response predictions to targeted therapies
- Understanding of pharmacodynamics and biologically relevant dosing and scheduling
Our RPPA service
Service deliverables
Validation with Western blot
Validation with Western blot
- Spreadsheet containing sample identifiers and quantitated expression levels of each target proteins;
- Each dataset includes raw, normalized, and median-centered data, along with a pair of heatmaps (one supervised and one un-supervised)
- A study report.
Validation with Western blot
Validation with Western blot
Validation with Western blot
- The above figure shows a comparative analysis of AKT phosphorylation in cancer cell lines, utilizing both RPPA and Western blot techniques. Lysates from 14 cell lines cultured in standard complete media were subjected to analysis using RPPA and Western blot methods. The results are presented as relative fluorescence intensity (RFI) values displayed in a bar chart, alongside the corresponding raw western blot images.
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