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 microM 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 microM 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.
Brueggemeier
Shawn B
SB
Department of Chemical and Biological Engineering, University of Wisconsin-Madison, 1415 Engineering Drive, Madison, WI 53706, USA.
Kron
Stephen J
SJ
Palecek
Sean P
SP
D000180:Acrylic Resins; D004800:Enzymes, Immobilized; D005898:Glass; D005982:Glutathione Transferase; D020100:Hydrogels; D011506:Proteins / Q000032:analysis* / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D000099166:Polyacrylamides
D000180:Acrylic Resins; D004800:Enzymes, Immobilized; D005982:Glutathione Transferase; D020100:Hydrogels; D011506:Proteins; D011993:Recombinant Fusion Proteins; D000099166:Polyacrylamides