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.sup.2) was detectable by direct measurement of green
fluorescent protein fluorescence and this lower detection limit was
reduced to 0.080 ng/mm.sup.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.