Use of protein–acrylamide copolymer hydrogels for measuring protein concentration and activity

We report the development and characterization of a polyacrylamide-based protein immobilization strategy for surface-bound protein assays, including concentration detection, binding affinity, and enzyme kinetics. Glutathione S-transferase (GST) fusion proteins have been labeled with an acrylic moiety and attached to acrylic-functionalized glass surfaces through copolymerization with acrylic monomer. The specific attachment of GST–green fluorescent protein (GFP) fusion protein was more than sevenfold greater than the nonspecific attachment of nonacrylic-labeled GST–GFP; 0.32 ng/mm 2 of surface-attached GST–GFP was detectable by direct measurement of GFP 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. Michaelis–Menten kinetic constants for the reaction occurring in solution were K m =2.7±1.0 μ M and V max=8.1±3.1 (arbitrary units). Kinetic values for the reaction utilizing surface-immobilized substrate were K m =0.36±0.033 μ M and V max=9.7±0.63 and were found to be independent of the acrylamide concentration within the copolymer. Such a surface attachment strategy should be applicable to the proteomics field and addresses denaturation and dehydration problems associated with protein microarray development.