Protein Arrays III: Reverse-Phase Protein Arrays
The reverse-phase protein array (RPPA) is to use highly specific antibodies to interrogate pan or posttranslationally modified protein targets, such as phosphorylated proteins, particularly the proteins involved in cell signaling pathways. In this protocol we will cover the preparation of cell (or tissue) lysates, sample printing, antibody validation, antibody interrogation, signal amplification steps, imaging and data analysis. In this protocol, colorimetric catalyzed signal amplification (CSA) chemistry, fluorescence and near-infrared (NIR) based detection methods will be described.
Key wordsReverse-phase protein array Antibody validation Cell signaling pathways Near-infrared (NIR) fluorescence
This work was partly supported by a grant from the Lupus Research Institute to T.W. and a startup fund from the University of Houston to T.W.
- 2.Tibes R, Qiu Y, Lu Y, Hennessy B, Andreeff M, Mills GB, Kornblau SM (2006) Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells. Mol Cancer Ther 5(10):2512–2521. doi: 10.1158/1535-7163.MCT-06-0334 CrossRefPubMedGoogle Scholar
- 3.Sheehan KM, Calvert VS, Kay EW, Lu Y, Fishman D, Espina V, Aquino J, Speer R, Araujo R, Mills GB, Liotta LA, Petricoin EF 3rd, Wulfkuhle JD (2005) Use of reverse phase protein microarrays and reference standard development for molecular network analysis of metastatic ovarian carcinoma. Mol Cell Proteomics 4(4):346–355. doi: 10.1074/mcp.T500003-MCP200 CrossRefPubMedGoogle Scholar
- 5.Carter BZ, Qiu Y, Huang X, Diao L, Zhang N, Coombes KR, Mak DH, Konopleva M, Cortes J, Kantarjian HM, Mills GB, Andreeff M, Kornblau SM (2012) Survivin is highly expressed in CD34(+)38(−) leukemic stem/progenitor cells and predicts poor clinical outcomes in AML. Blood 120(1):173–180. doi: 10.1182/blood-2012-02-409888 CrossRefPubMedPubMedCentralGoogle Scholar
- 7.Lui VW, Peyser ND, Ng PK, Hritz J, Zeng Y, Lu Y, Li H, Wang L, Gilbert BR, General IJ, Bahar I, Ju Z, Wang Z, Pendleton KP, Xiao X, Du Y, Vries JK, Hammerman PS, Garraway LA, Mills GB, Johnson DE, Grandis JR (2014) Frequent mutation of receptor protein tyrosine phosphatases provides a mechanism for STAT3 hyperactivation in head and neck cancer. Proc Natl Acad Sci U S A 111(3):1114–1119. doi: 10.1073/pnas.1319551111 CrossRefPubMedPubMedCentralGoogle Scholar