Protein and Antibody Array Proteomics
(Shixia Huang, Ph.D., Director)
Baylor College of Medicine 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 provide 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. The Luminex web site can be checked for more information.
- Antibody/Protein arrays
Antibody arrays have recently been developed by several vendors (see table below) that provide sensitive and specific protein marker discovery tools. These arrays have as many as 308-1318 antibodies on a single slide that collectively covers >2000 unique proteins, the majority of which are low abundance regulatory proteins important in cancer biology. Antibodies in these arrays are against total as well as phosphorylation states of certain proteins.
|Vendor||Functional groups||Number of antibodies|
|Sigma Gene||regulation, cell signaling, MAPK & PKC pathways, p53 pathways||725|
|RayBiotech||Cytokines, adipokines, growth factors, chemokines, MMPS, Phosphoproteins||507 human 308 mouse|
|Full Moon BioSystems||Intracellular pathways: AKT, MAPK, NF-kB, JAK/STAT; nuclear and membrane receptors, tyrosine kinases, cancer/apoptosis, cell cycle, chromatin/transcription, cytoskeleton, neuroscience, insulin signaling||656|
|Full Moon BioSystems||Phosphor-specific antibody array||1318|
For Sigma arrays, proteins are extracted from two different samples, each is labeled with fluorescent Cy3 or Cy5 dyes, and equal amount of bound proteins from the two different samples are combined to react with antibodies on the same slide (two color arrays). The RayBiotech system and Full Moon BioSystems arrays detect proteins in a single sample labeled with biotin and uses fluorescent-conjugated streptavidin as the detection probe (one-color array). Antibody arrays are scanned using a GenePix 4200AL scanner from Molecular Devices.
We are in the process of optimizing and validating commercially available arrays for protein-protein interaction, including Invitrogen’s protoarray with >9,200 proteins and Panomics’s protein arrays directed to specific sets of signaling proteins.
- Reverse Phase Protein Arrays
The Reverse-Phase Protein Array (RPPA) serves to analyze specific pathways and their activation states. This technology is ideal for comparisons of post-translationally modified proteins and their total protein counterparts. The platform has been validated for cells and tissue samples and we are currently evaluating serum and body fluids. Using a limited amount of sample for analysis, a unique profile can be created using over 170 high-quality antibodies. This platform is ideal for large sample sets but can yield a wealth of information for small sample sets due to its ability to use a minute amount of sample. Protein samples are extracted from cells and tissue samples and denatured. Samples are printed in triplicate onto specialized nitrocellulose-coated slides using an Aushon 2470 Arrayer. Appropriate controls are printed alongside the samples. Samples are then probed with different antibodies and labeled with fluorescent dye using a Dako Autostainer Link48. Samples are visualized using a Molecular Devices GenePix 4200 AL scanner.
- 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 Biostatistics and Informatics shared resource (Directors: Dr. Susan Hilsenbeck and Dr. Lauren Becnel).
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 (5 or 4 or others) logistic-fitting method is selected based on the available points of quality standards. Quantitation of the data, with CV% of replicates, is calculated based on standard curves. Data are then exported to excel spreadsheets for investigators.
GenePix Pro software is used for antibody array 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 is performed by our statistician or in the conjunction to the Biostatistics and Informatics shared resource (Directors: Dr. Susan Hilsenbeck and Dr. Lauren Becnel). The Proteomics Core provides a full service which includes performing data analysis for the RPPA platform. After the samples are processed, the data is normalized and analyzed. For each antibody, a mean intensity signal of each sample is generated. The corresponding negative control signal intensity is subtracted from it. The resulting signal is then normalized to its total protein staining value. Analysis is then performed using the R statistical software (http://www.r-project.org).
Charge Back Fees and Service Request Forms
Fees: Please, see the charge back fees list for detailed price estimates and examples.
Service Request Form: Please, fill out the service request form for project/service inquiry.
Shixia Huang, Ph.D., Director
Associate Professor, Department of Molecular & Cellular Biology
Myra Custorio, B.S.
Sr. Research Assistant,
Baylor College of Medicine
One Baylor Plaza, Margaret M. Alkek Research Building, R507/R551C
Houston, TX 77030