| abstract |
Polyacrylamide-based methods of fabricating surface-bound peptide and protein arrays, the arrays themselves, and a method of using the arrays to detect biomolecules and to measure their concentration, binding affinity, and kinetics are described. Peptides, proteins, fusion proteins, protein complexes, nucleic acids, and the like, are labeled with an acrylic moiety and attached to acrylic-functionalized glass surfaces through a copolymerization with acrylic monomer. The specific attachment of glutathione S-transferase-green fluorescent protein (GST-GFP) fusion protein was more than 7-fold greater than the nonspecific attachment of non-acrylic labeled GST-GFP. Surface-attached GST-GFP (0.32 ng/mm 2 ) was detectable by direct measurement of green fluorescent protein fluorescence and this lower detection limit was reduced to 0.080 ng/mm 2 using indirect antibody-based detection. The polyacrylamide-based surface attachment strategy was also used to measure the kinetics of substrate phosphorylation by the kinase c-Src which is encoded by the Rous Sarcoma virus. The surface attachment strategy is applicable to the proteomics field and addresses denaturation and dehydration problems associated with protein microarray development. |