Abstract
There are many instances in which it is desirable to generate profiles of the relative abundance of a multiplicity of protein species. Examples include studies in embryonic development, immunobiology, drug responses, cancer biology, biomarkers, and so on. Microarray formats provide a convenient, high-throughput vehicle for generating such profiles, and the repertoire of proteins that can be measured is growing continuously as larger panels of specific antibodies become available. Here we describe methods for the use of antibody microarrays, whereby the detection of specifically bound antigens is enhanced by rolling circle amplification (RCA). RCA-enhanced protein detection on antibody microarrays provides a means for rapid protein profiling at high sensitivity. The set of RCA reagents remains unchanged for different microarray formats and compositions, and signal readout is performed using standard fluorescent dyes and scanners. The method is sensitive enough for the most challenging applications, such as the detection of low-abundance components of human serum.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Haab B. B., Dunham M. J., and Brown P. O. (2001) Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions. Genome Biol. 2, 1–13.
MacBeath G. and Schreiber S. L. (2000) Printing proteins as microarrays for highthroughput function determination. Science 289, 1760–1763.
Schweitzer B., Roberts S., Grimwade B., et al. (2002) Multiplexed protein profiling on microarrays by rolling-circle amplification. Nat. Biotechnol. 20, 359–365.
Knezevic V., Leethanakul C., Bichsel V. E., et al. (2001) Proteomic profiling of the cancer microenvironment by antibody arrays. Proteomics 1, 1271–1278.
Sreekumar A., Nyati M. K., Varambally S., et al. (2001) Profiling of cancer cells using protein microarrays: discovery of novel radiation-regulated proteins. Cancer Res. 61, 7585–7593.
Huang R-P., Huang R., Fan Y., and Lin Y. (2001) Simultaneous detection of multiple cytokines from conditioned media and patient’s sera by an antibody-based protein array system. Anal. Biochem. 294, 55–62.
Miller J. C., Zhou H., Kwekel J., et al. (2003) Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers. Proteomics 3, 56–63.
Nielsen U. B., Cardone M. H., Sinskey A. J., MacBeath G., and Sorger P. K. (2003) Profiling receptor tyrosine kinase activation by using Ab microarrays. Proc. Natl. Acad. Sci. USA 100, 9330–9335.
Huang R., Lin Y., Wang C. C., et al. (2002) Connexin 43 suppresses human glioblastoma cell growth by down-regulation of monocyte chemotactic protein 1, as discovered using protein array technology. Cancer Res. 62, 2806–2812.
Lin Y., Huang R., Santanam N., Liu Y. G., Parthasarathy S., and Huang R. P. (2002) Profiling of human cytokines in healthy individuals with vitamin E supplementation by antibody array. Cancer Lett. 187, 17–24.
Tannapfel A., Anhalt K., Hausermann P., et al. (2003) Identification of novel proteins associated with hepatocellular carcinomas using protein microarrays. J. Pathol. 201, 238–249.
Nallur G., Luo C., Fang L., et al. (2001) Signal amplification by rolling circle amplification on DNA microarrays. Nucleic Acids Res. 29, E118.
Lizardi P. M., Huang X., Zhu Z., Bray-Ward P., Thomas D. C., and Ward D. C. (1998) Mutation detection and single-molecule counting using isothermal rollingcircle amplification. Nat. Genet. 19, 225–232.
Ladner D. P., Leamon J. H., Hamann S., et al. (2001) Multiplex detection of hotspot mutations by rolling circle enabled universal microarrays. Lab. Invest. 81, 1079–1086.
Schweitzer B., Wiltshire S., Lambert J., et al. (2000) Imunoassays with rolling circle DNA amplification: a versatile platform for ultrasensitive antigen detection. Proc. Natl. Acad. Sci. USA 97, 10,113-10,119.
Zhou H., Bouwman K., Schotanus M., et al. (2004) Two-color, rolling-circle amplification on antibody microarrays for sensitive, multiplexed serum-protein measurements. Genome Biol. 5, R28.
Guschin D., Yershov G., Zaslavsky A., et al. (1997) Manual manufacturing of oligonucleotide, DNA, and protein microchips. Anal. Biochem. 250, 203–211.
Arenkov P., Kukhtin A., Gemmell A., Voloshchuk S., Chupeeva V., and Mirzabekov A. (2000) Protein microchips: Use for immunoassay and enzymatic reactions. Anal. Biochem. 278, 123–131.
Mathies R. A., Peck K., and Stryer L. (1990) Optimization of high-sensitivity fluorescence detection. Anal. Chem. 62, 1786–91.
Nielsen U. B. and Geierstanger B. H. (2004) Multiplexed sandwich assays in microarray format. J. Immunol. Methods 290, 107–120.
Leung Y. F. and Cavalieri D. (2003) Fundamentals of cDNA microarray dataanalysis. Trends Genet. 19(11), 649–659.
Bolstad B. M., Collin F., Simpson K. M., Irizarry R. A., and Speed T. P. (2004) Experimental design and low-level analysis of microarray data. Int. Rev. Neurobiol. 60, 25–58.
Ochs M. F., Moloshok T. D., Bidaut G., and Toby G. (2004) Bayesian decomposition: analyzing microarray data within a biological context. Ann. NY Acad. Sci. 1020, 212–226.
Park T., Yi S. G., Kang S. H., Lee S., Lee Y. S., and Simon R. (2003) Evaluation of normalization methods for microarray data. BMC Bioinformatics 4, 33.
Yang Y. H., Dudoit S., Luu P., Lin D. M., Peng V., Ngai J., and Speed T. P. (2002) Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30, e15.
Fare T. L., Coffey E. M., Dai H., et al. (2003) Effects of atmospheric ozone on microarray data quality. Anal. Chem. 75, 4672–4675.
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Haab, B.B., Lizardi, P.M. (2006). RCA-Enhanced Protein Detection Arrays. In: New and Emerging Proteomic Techniques. Methods in Molecular Biology™, vol 328. Humana Press. https://doi.org/10.1385/1-59745-026-X:15
Download citation
DOI: https://doi.org/10.1385/1-59745-026-X:15
Publisher Name: Humana Press
Print ISBN: 978-1-58829-519-4
Online ISBN: 978-1-59745-026-3
eBook Packages: Springer Protocols