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The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers
Published on: January 22, 2013
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Protein Arrays III: Reverse-Phase Protein Arrays.
Yulin Yuan1,2, Xia Hong1,3, Zuan-Tao Lin1
1Department of Biomedical Engineering, University of Houston, 3605 Cullen Blvd, Houston, TX, 77204-5060, USA.
Methods in Molecular Biology (Clifton, N.J.)
|October 8, 2017
Summary
Reverse-phase protein arrays (RPPA) utilize specific antibodies to analyze protein targets, including those involved in cell signaling. This protocol details RPPA methods from sample preparation to data analysis, covering various detection techniques.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Reverse-phase protein array (RPPA) is a powerful technique for high-throughput proteomic analysis.
- RPPA enables the interrogation of specific protein targets, including post-translationally modified proteins like phosphoproteins.
- Understanding cell signaling pathways relies on accurate protein quantification and analysis.
Purpose of the Study:
- To provide a comprehensive protocol for performing Reverse-Phase Protein Arrays (RPPA).
- To detail the steps involved in sample preparation, antibody validation, and data analysis for RPPA.
- To describe various detection methods including colorimetric catalyzed signal amplification (CSA), fluorescence, and near-infrared (NIR).
Main Methods:
- Preparation of cell or tissue lysates.
- Sample printing onto specialized slides.
- Antibody validation and interrogation.
- Signal amplification using CSA, fluorescence, or NIR detection.
- Imaging and data analysis.
Main Results:
- The protocol successfully outlines the complete workflow for RPPA.
- Different detection methods (CSA, fluorescence, NIR) are described for signal visualization.
- The protocol facilitates the analysis of protein expression and modification states.
Conclusions:
- This protocol provides a standardized methodology for RPPA implementation.
- The described techniques enable robust analysis of protein targets in cell signaling.
- RPPA is a versatile tool for quantitative proteomic studies.
