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We have developed a number of methods for immobilizing ligands with control over the densities and patterns of each ligand. We first developed a Diels-Alder mediated scheme for immobilizing ligands.43 This reaction was important because it offered quantitative control over the densities of immobilized ligands, a benefit that derives from the well-behaved kinetics for the reaction and the ability to use electrochemical techniques to quantitate the amount of ligand on the substrate.46 This issue of density is especially important in studying the polyvalent adhesion of cells.48 We have also adapted the maleimide-mediated immobilization of thiol-tagged ligands.70 These two methods have proven quite general and useful for immobilizing peptides,61 carbohydrates,64 and other low molecular weight ligands but they are not suited to immobilizing recombinant proteins. For this latter need, protein immobilization methods that use recombinantly tagged proteins (GST, oligoHis) benefit from affinity capture of the protein to the surface with uniform control over orientation but suffer from poor stability over long times because of the reversible nature of the binding interaction. We developed an analogous method that employs cutinase as a fusion tag and uses monolayers that present a phosphonate ligand that is an irreversible inhibitor of the cutinase active site.66 A fusion protein of cutinase and the tenth type III repeat from fibronectin underwent efficient immobilization and supported cell adhesion.80 This methodology extends our methods for immobilization from low molecular weight ligands to recombinant proteins, and therefore extends the complexity of the model substrates.
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