Submillimeter-scale surface gradients of immobilized protein ligands.
We describe a method to produce antibody-captured ligand gradients over biologically relevant distances (hundreds of micrometers) whereby the ligand density and gradient shape may be tailored. Separation of the ligand from the solid-phase 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 (Dll1). To visualize the gradient of Dll1, we have used the multistep approach of binding with rabbit anti-Dll1 primary antibody and then adding colloidal-gold-conjugated secondary antibody.
Walker
Robert A
RA
MRC Centre for Developmental and Biomedical Genetics, Department of Biomedical Science, Firth Court, Western Bank, Sheffield S10 2TN, UK.
Cunliffe
Vincent T
VT
Whittle
Jason D
JD
Steele
David A
DA
Short
Robert D
RD
D000179:Acrylates / Q000737:chemistry; D000906:Antibodies / Q000737:chemistry; D001672:Biocompatible Materials; D002135:Calcium-Binding Proteins; D002234:Carbodiimides / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D005898:Glass; D006358:Hot Temperature; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry; D011312:Pressure; D011506:Proteins / Q000737:chemistry*; D015398:Signal Transduction; D013499:Surface Properties
D000179:Acrylates; D000906:Antibodies; D001672:Biocompatible Materials; D002135:Calcium-Binding Proteins; D002234:Carbodiimides; C512941:DLK1 protein, human; D005456:Fluorescent Dyes; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D008565:Membrane Proteins; D011506:Proteins; C036658:acrylic acid