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 260+ 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 have also developed an epigenetic RPPA platform for histone marks and chromatin modifying 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 have developed in-house normalization and statistical analyses algorithms.
Epigenetic RPPA for chromatin modifier proteins use whole cell lysate extract and are routinely profiled with our above mentioned RPPA panels. Epigenetic RPPA for Histone marks require purified histone and is assayed separated.
Learn more about RPPA technology and services.
Multiplex Quantitative Analyses Using the Suspension Array System (Bio-Plex From Bio-Rad) Through Luminex xMAP Bead Technology
The Luminex xMAP technology is a microsphere-based multiplexing system for quantitation of multiple analytes in a single sample. A specific antibody to the target protein is covalently coupled to internal fluorescently dyed beads. The beads with bound target proteins are separated by laser excitation and quantitated. Beads coupled with different antibodies, each with a distinct fluorescence signature, are mixed, thus enabling simultaneous assay of multiple protein targets in a single well of a 96-well plate. This is a sensitive quantitative assay that enables detection of proteins in cell extracts and extra-cellular fluids at very low concentrations (1 pg/ml), and thus is excellent for analysis of signaling pathways and regulatory proteins. We currently support a Luminex 200 (Bio-Plex) instrument and have optimized and provided Luminex assays for cytokines, growth factors, apoptosis, cancer, metabolic markers and Akt and MAP kinase signaling pathways. As commercial vendors expand the range of Luminex beads, we will validate assays for new bead kits as they become available. For more information, see the Luminex website.
We offer a range of Luminex services to investigators from full service to independent users. In Full Service assays, investigators provide the samples and the assay and analysis is performed by the Proteomics Core. For investigators who prefer to perform the assays themselves, our Luminex instrument and software can be used for a fee to read the assays and perform analysis.
Data Analysis Consultation
Preliminary analysis of the data generated by these protein profiling platforms is provided as a service using software packages provided by the vendors. For more sophisticated statistical analysis, projects are directed to the Multi-Omics Data Analysis. (Director: Dr. Cristian Coarfa, Ph.D.).
Bio-Plex Manager Software Station
The Bio-Plex Manager Software station is available for analysis of Luminex data. Quality control samples are included to assess each analyte and a specific parameter (five or four or others) logistic-fitting method is selected based on the available points of quality standards. Quantitation of the data, with CV percent of replicates, is calculated based on standard curves. Data are then exported to excel spreadsheets for investigators.
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 deferentially 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.