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 360+ 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, nuclear receptors/transcriptional, chromatin regulatory, and metabolism 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 an epigenetic RPPA platform to measure histone modifications. We have developed in-house normalization and statistical analyses algorithms.
Multiplex Quantitative Analyses Using the Suspension Array System (Luminex) 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 (and Bio-Plex 200) instrument and have optimized and provided Luminex assays for cytokines, growth factors, apoptosis, cancer, metabolic markers and Akt and MAP kinase signaling pathways. Commercial vendors are constantly expanding the range of Luminex beads, so please inquire about bead kit availability. For more information, see the Luminex website.
We offer a range of Luminex services to investigators from plate reading to full service. 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.
Ella Automated Immunoassay System
ELLA is an automated immunoassay platform that performs assays similar to ELISA, but with the advantage of requiring smaller sample materials and incorporating automation. This system can detect up to sub-picogram level sensitivity, has the ability to multiplex up to 8 targets per sample. It complements the Luminex multiplex immunoassay platform. Possible applications for Ella include cell and gene therapy, bioprocessing, neuroscience, cancer, and immunology.
Ella is a cartridge-based microfluidic platform for quickly and reliably measuring many clinical or laboratory samples at once. Microfluidic channels contain 3 glass nanoreactors (GNRs) coated with capture antibody, generating triplicate data with every run. Ella utilizes a robust amplification process involving biotin-streptavidin, followed by fluorescence labeling, and enhances sensitivity through laser camera capture of signals. After diluting and loading samples and wash buffer, the cartridge, containing all other necessary reagents for the immunoassay, is ready to run in the Ella machine. Ella performs the incubation, washing, and detection steps, eliminating manual steps and increasing reproducibility.
For more information, please visit the Ella automated immunoassay website.
Reference: Wessels U, Mackeben K, Stubenrauch K. Use of Ella® to facilitate drug quantification and antidrug antibody detection in preclinical studies. Bioanalysis. 2019 Feb;11(3):153-164. doi: 10.4155/bio-2018-0288. Epub 2019 Jan 10. PMID: 30628464.
OLINK Reveal for 1000+ Protein Markers
Olink Reveal is an NGS-based proteomics platform using Proximity Extension Assay (PEA) technology, combining dual antibody specificity with DNA barcoding and sequencing readout for high sensitivity and scalability. It enables simultaneous profiling of >1,000 proteins from serum, plasma, and other sample types, including tissue and cell lysates.
The panel provides strong coverage of immune and inflammatory signaling, along with key components of oncogenic and signaling pathways, including PI3K–AKT, MAPK, receptor tyrosine kinase signaling, NF-κB, interferon responses, and cellular stress pathways. It also includes markers of apoptosis and necroptosis (e.g., RIPK3, GSDMD), enabling pathway-level analysis across immune and cancer biology.
This platform is complementary to RPPA, which provides post-translational modification and phospho-signaling measurements, together enabling integrated profiling of circulating proteins and intracellular signaling activity.
Please see the full antibody list.
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.