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Reverse-Phase Protein Lysate Microarrays
Reverse-phase Protein Lysate microarrays (RPAs) provide an exciting new platform for measuring protein expression levels
in a large number of biological samples simultaneously. RPA’s, originally introduced by Dr. Lance Liotta of the National Institutes
of Health and Dr. Emanuel Petricoin of the FDA, are essentially large arrays of micro dot-blots. They can provide accurate, sensitive
and quantitative protein expression data on many samples in a single experiment. Proteomic profiling using RPAs yields more direct
answers to functional and pharmacological questions than transcriptional profiling. Dr. Liotta and Dr. Petrcoin are presently co-Directors
of the Center for Applied Proteomics and Molecular Medicine at George Mason University.
RPAs were first described by Paweletz et al. to measure subtle quantitative changes in multiple classes of proteins from individual
cell types within tissues. Since the initial publication, this technology has also been applied to other biological materials.
Today, there are several notable uses for RPAs such as protein monitoring for bio-marker discovery and the monitoring of signal
transduction proteins in response to various biological stimuli. For example, government researchers and pharmaceutical companies are
using RPAs to map protein signaling pathways, assess drug target and biomarker expression, and understand a drug candidate’s mechanism
of action. Dr. Nishizuka (Nishizuka et al.) of the National Institutes of Health has created 10,000-element lysate arrays for pathway
mapping, thereby permitting detailed time-course studies to be performed of protein response to cell stimulation. The ability to
quantitatively track, for example, the phosphorylation cascade down one branch of a pathway versus another can guide researchers in
developing compounds that best elicit the desired therapeutic effects.
Clinical researchers are also examining RPAs as a potential early means of implementing personalized cancer treatment (Espina et al.).
By examining the expression levels of key proteins in cancer cells isolated via laser-capture microdissection, researchers can identify the
exact nature of protein mis-expression. The goal of this work is to allow clinicians to recommend therapy tuned to the exact nature of a
patient’s cancer.
One of the most sensitive detection methodologies for RPAs involves colorimetric detection via amplification steps involving tagged
secondary antibodies. The RPA data, viewed as series of dilution curves, is a sensitive, quantitative, and much higher throughput alternative
to Western blotting. Aushon BioSystems’ 2470 arrayer is a proven technology for building exceptionally high quality Reverse-phase Protein
Lysate microarrays (RPA). The 2470 can produce arrays of the often viscous lysate material with the linearity and consistency to permit
data extraction from dilution series curves.
Links:
Molecular Translational Technologies MTP/CCR/NCI/NIH
GMU Center for Applied Proteomics and Molecular Medicine
References:
- Paweletz et al. 2001. Oncogene 20: 1981-1989
- Espina et al. 2003. Proteomics 3: 2091-2100
- Nishizuka et al. 2003a. PNAS 100: 14229-14234
- Nishizuka et al. 2003b. Cancer Research 63: 5243-5250
- Nishizuka et al. 2006. BioTechniques 40(4): 442-448
- Nishizuka et al., 2006, European Journal of Cancer
- Wulfkuhle et al., 2006, Vol 3 No 5 Nature Clinical Practice Oncology
- Petricoin et al., 2007, Cancer Res; 67: (7)
- Shankavaram et al., 2007, Molecular Cancer Therapeutics: 6(3) 820-832
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