Reverse Phase Protein Arrays
Reverse phase protein array is a high-throughput technology that performs protein assays on thousands of samples simultaneously, including tissue and cell lysates, serum, plasma or other body fluids. This protein array platform measures levels of protein expression, as well as protein modifications such as phosphorylation. Protein lysates are arrayed as microspots on nitrocellulized coated glass slides and probed with highly specific antibodies that have been validated for RPPA. Each microspot contains the whole proteome repertoire of the tissue or cell. This enables the determination of a set of parameters in large collections of tissue or cell samples.
We currently have an inventory of 240+ validated antibodies that cover multiple total proteins and phosphoproteins in the following protein pathways or functional protein groups: epithelial-mesenchymal transition (EMT), stem cells, apoptosis, DNA damage, autophagy, proliferation and cell cycle, growth factor receptors, cytokines/STATs and nuclear receptors/transcriptional and chromatin regulatory proteins. We continuously work together with BCM investigators to build RPPA assays for new protein pathways of interest and to validate the required antibodies. We are also working to be able to do analysis with various types of samples including cell/tissue lysates, macro-dissected tumor samples, laser capture micro-dissected (LCM) samples, small numbers of isolated stem cells or other rare cell types, and body fluids such as serum and urine. A novel application we have developed is to analyze isolated protein complexes. We are also developing an epigenetic RPPA platform to measure histone modifications. We have developed in-house normalization and statistical analyses algorithms.
GenePix Pro Software
GenePix Pro Software is used for RPPA image analysis to coordinate the spots on the array with the protein information file, and to perform accurate alignment, adjust the size of each spot, and exclude spots with bad quality. Result files are generated with background intensity, signal intensity, and signal to noise ratios. The result file is also used for further normalization and statistical analyses to identify differentially expressed proteins among different samples.
Analysis for the Reverse Phase Antibody Array Platform
Analysis for the Reverse Phase Antibody Array platform is performed by our statistician in the conjunction to the Multi-Omics Data Analysis. (Director: Dr. Cristian Coarfa, Ph.D.). We provide a full service which includes image analysis, data normalization and processing for the RPPA platform. After the samples are processed, the data is normalized and analyzed. For each antibody, a median intensity signal of each spot is generated. The corresponding negative control signal intensity is subtracted from it. The resulting signal is then normalized to the total protein staining value and ready for statistical analysis.
CPRIT Cancer Proteomics and Metabolomics
Additional services are available through CPRIT Cancer Proteomics and Metabolomics - a group funded through a grant from the Cancer Prevention Research Institute of Texas.