Summary
We have discovered a novel protein immobilization method, i.e., a “Three-Dimensional Nanostructured Protein Hydrogel” (3-D NPH), which is composed of protein-reactive polymer hybrid nanoparticles to detect protein–protein interactions. The 3-D NPH can be easily prepared by spotting a protein/reactive polymer mixture on a substrate.The resulting 3-D NPH is characterized by large amounts of immobilized proteins and a novel porous structure.
The 3-D NPH technology was applied to immobilize streptavidin (SA) onto Au-coated surface for surface plasmon resonance imaging (SPRi). By using 3-D NPH method, it was possible to improve the sensitivity of protein–protein interactions drastically comparing to the conventional protein immobilization method.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Schreiber, S. L., (1998) Chemical genetics resulting from a passion for synthetic organic chemistry. Bioorg. Med. Chem., 6, 1127–1152.
Haggarty, S. J., Mayer, T. U., Miyamoto, D. T., Fathi, R., King, R. W., Mitchison, T. J., and Schreiber, S. L. (2000) Dissecting cellular processes using small molecules: identification of colchicine-like, taxol-like and other small molecules that perturb mitosis. Chem. Biol., 7, 275–286.
Koga, H. (2006) Establishment of the platform for reverse chemical genetics targeting novel protein–protein interactions. Mol. Biosyst. 2, 159–164.
Spring, D. R., (2005) Chemical genetics to chemical genomics: small molecules offer big insights. Chem. Soc. Rev. 34, 472–482.
Macbeath, G., and Schreiber, S. L. (2000) Printing proteins as microarray for high throughput function determination. Science 289, 1760–1763.
Stiffler, M. A., Grantcharava, V. P., Sevecka, M., and MacBeath, G. (2006) Uncovering quantitative protein interaction networks for mouse PDZ domains using protein microarrays. J. Am. Chem. Soc. 128, 5913–5922.
Michaud, G. A., Salcius, M., Zhou, F., Bangham, R., Bonin, J., Guo, H., Snyder, M., Predki, P. F., and Schweitzer, B. I. (2003) Analyzing antibody specificity with whole proteome microarrays. Nat. Biotechnol. 21, 1509–12.
Zhu, H., and Snyder, M. (2003) Protein chip technology. Curr Opin Chem Biol, 7, 55–63.
Lofas, S., and Johnsson, B. (1990) A novel hydrogel matrix on gold surfaces in surface plasmon resonance sensors for fast and efficient covalent immobilization of ligands. J. Chem. Soc. Chem. Commun., 21, 1526–1528.
Polzius, R., Schneider, Th., Bier, F. F., Biltewski, U., and Koscinski, W. (1996) Optimization of biosensing using grating couplers: immobilization on tantalum oxide waveguides. Biosens. Bioelectron., 11, 503–514.
Arenkov, P., Kukhtin, A., Gemmell, A., Voloschchuk, S., Chupeeva, V., and Mirzabekov, A. (2000) Protein microchips: Use for immunoassay and enzymatic reactions. Anal. Biochem., 278, 123–131.
Angenendt, P., Glokler, J., Murphy, D., Lehrach, H., and Cahill, D. J., (2002) Toward optimized antibody microarrays: a comparison of current microarray support materials. Anal. Biochem., 309, 253–260.
Charles, P. T., Taitt, C. R., Goldman, E. R., Rangasammy, J. G., and Stenger, D. A. (2004) Immobilization strategy and characterization of hydrogel-based thin films for integration of ligand binding with staphylococcal enterotoxin B (SEB) in a protein microarray format. Langmuir, 20, 270–272.
Brueggemeier, S. B., Wu, D., Kron, S. J., and Palecek, S. P. (2005) Protein-acrylamide copolymer hydrogels for array-based detection of tyrosine kinase activity from cell lysates. Biomacromolecules, 6, 2765–2775.
Afanassiev, V., Hanemann, V., and Wolfl, S. (2000) Preparation of DNA and protein micro arrays on glass slides coated with an agarose film. Nucleic Acids Res., 28, 66e.
Dominguez, M. M., Wathier, M., Grinstaff, M. W., and Schaus, S. E. (2007) Immobilized Hydrogels for Screening of Molecular Interactions. Anal. Chem., 79, 1064–1066.
Dai, J., Bao, Z., Sun, L., Hong, S. U., Baker, G. L., and Bruening, M. L. (2006) High-capacity binding of proteins by poly(acrylic acid) brushes and their derivatives. Langmuir, 22, 4274–4281.
Y. Zhou, O. Andersson, P. Lindberg and Liedberg, B. (2004) Protein microarrays on carboxymethylated dextran hydrogels: immobilization, characterization and application. Microchim. Acta., 147, 21–30.
Tanaka, H., Shiroya, T., Hanasaki, M., Isojima, T., Takeuchi, H., and Kawaguchi, H., (2008) Preparation of porous protein-based hydrogel for highly sensitive protein chips. Macromol. Symp., 266, 81–84.
Tanaka, H., Isojima, T., Hanasaki, M., Ifuku, Y., Takeuchi, H., Kawaguchi, H., and Shiroya, T. (2008) Porous protein-based nanoparticle hydrogel for protein chips with improved sensitivity. Macromol. Rapid Commun., 29, 1287–1292.
D’Agosto, F., Charreyre, M.-T., Melis, F., Mandrand, B., and Pichot, C., (2003) High molecular weight hydrophilic functional copolymers by free-radical copolymerization of acrylamide and N-acryloylmorpholine with N-acryloxysuccinimide: Application to the synthesis of a graft copolymer. J. Appl. Polym. Sci., 88, 1808–1816.
Habauzit, D., Chopineau, J., and Roig, B. (2007) SPR-based biosensors: a tool for biodetection of hormonal compounds. Anal. Bioanal. Chem., 387, 1215–1223.
Stenberg, E., Persson, B., Roos, H., and Urbaniczky, C. (1991) Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins. J. Colloid Interface Sci., 143, 513–526.
Acknowledgments
We thank Dr. Hisashi Koga of Kazusa DNA Research Institute and Haruma Kawaguchi of Keio University for their helpful suggestions.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Shiroya, T., Tanaka, H., Hanasaki, M., Takeuchi, H. (2009). Preparation of Highly Sensitive Protein Array Using Reactive Polymer. In: Koga, H. (eds) Reverse Chemical Genetics. Methods in Molecular Biology™, vol 577. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-232-2_16
Download citation
DOI: https://doi.org/10.1007/978-1-60761-232-2_16
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-231-5
Online ISBN: 978-1-60761-232-2
eBook Packages: Springer Protocols