TY - JOUR
T1 - Reverse-phase protein array
T2 - Technology, application, data processing, and integration
AU - Coarfa, Cristian
AU - Grimm, Sandra L.
AU - Rajapakshe, Kimal
AU - Perera, Dimuthu
AU - Lu, Hsin Yi
AU - Wang, Xuan
AU - Christensen, Kurt R.
AU - Mo, Qianxing
AU - Edwards, Dean P.
AU - Huang, Shixia
N1 - Publisher Copyright:
© 2021 ABRF.
PY - 2021
Y1 - 2021
N2 - Reverse-phase protein array (RPPA) is a high-throughput antibody-based targeted proteomics platform that can quantify hundreds of proteins in thousands of samples derived from tissue or cell lysates, serum, plasma, or other body fluids. Protein samples are robotically arrayed as microspots on nitrocellulose-coated glass slides. Each slide is probed with a specific antibody that can detect levels of total protein expression or post-translational modifications, such as phosphorylation as a measure of protein activity. Here we describe workflow protocols and software tools that we have developed and optimized for RPPA in a core facility setting that includes sample preparation, microarray mapping and printing of protein samples, antibody labeling, slide scanning, image analysis, data normalization and quality control, data reporting, statistical analysis, and management of data. Our RPPA platform currently analyzes;240 validated antibodies that primarily detect proteins in signaling pathways and cellular processes that are important in cancer biology. This is a robust technology that has proven to be of value for both validation and discovery proteomic research and integration with other omics data sets.
AB - Reverse-phase protein array (RPPA) is a high-throughput antibody-based targeted proteomics platform that can quantify hundreds of proteins in thousands of samples derived from tissue or cell lysates, serum, plasma, or other body fluids. Protein samples are robotically arrayed as microspots on nitrocellulose-coated glass slides. Each slide is probed with a specific antibody that can detect levels of total protein expression or post-translational modifications, such as phosphorylation as a measure of protein activity. Here we describe workflow protocols and software tools that we have developed and optimized for RPPA in a core facility setting that includes sample preparation, microarray mapping and printing of protein samples, antibody labeling, slide scanning, image analysis, data normalization and quality control, data reporting, statistical analysis, and management of data. Our RPPA platform currently analyzes;240 validated antibodies that primarily detect proteins in signaling pathways and cellular processes that are important in cancer biology. This is a robust technology that has proven to be of value for both validation and discovery proteomic research and integration with other omics data sets.
KW - Post-translational
KW - Protein array analysis
KW - Proteomics
KW - Software
KW - Validation study
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UR - http://www.scopus.com/inward/citedby.url?scp=85106749031&partnerID=8YFLogxK
U2 - 10.7171/jbt.21-3202-001
DO - 10.7171/jbt.21-3202-001
M3 - Article
C2 - 33584151
AN - SCOPUS:85106749031
SN - 1524-0215
VL - 32
SP - 15
EP - 29
JO - Journal of Biomolecular Techniques
JF - Journal of Biomolecular Techniques
IS - 1
ER -