We describe a method to produce antibody-captured ligand gradients over biologically relevant distances (hundreds of micrcometers) whereby the ligand density and gradient shape may be tailored. Separation of the ligand from the solidphase surface ensures that the biological activity of the ligand remains unaffected by immobilization. Our method involves the use of a plasma-masking method to generate a surface chemical gradient on a glass substrate to which the 9E10 antibody is covalently coupled. This antibody captures myc-tagged biomolecules. In our example, the antibody is then used to immobilize a gradient of the intercellular signaling molecule delta-like-1 (DII1). To visualize the gradient of DII1, we have used the multistep approach of binding with rabbit anti-DII1 primary antibody and then adding colloidal-gold-conjugated secondary antibody.