A new approach to illustrate the principle of signal transduction and to assemble protein multilayers is described. It is based on competing affinities of two different ligands for the same binding site of a protein. A low-affinity ligand can be attached covalently to the protein, where it will be buried in the binding site and thus be prevented to interact with other proteins that recognize it. However, if a high-affinity ligand (or a molecule containing this ligand) is added, it will displace the low-affinity ligand (which still remains covalently bound) from the binding site to the periphery. The low-affinity ligand is now available for interaction with other molecules, thus providing the means through which to assemble multilayers of proteins by a "recognition cascade". This principle was demonstrated using the protein avidin which binds two ligands, biotin and 4-hydroxyazobenzene-2-carboxylic acid (HABA), with markedly different affinities. Avidin was affinity labeled with HABA, the low-affinity ligand, to produce a red, covalently conjugated avidin-HABA derivative (red avidin). Anti-HABA antibodies failed to recognize HABA buried in the binding site of avidin. However, upon addition of the high-affinity ligand biotin, HABA was expelled from the binding site and immediately bound by the antibodies. Multilayer assemblies of HABAylated avidin and biotinylated anti-HABA antibodies could thus be constructed. This concept may find application in numerous fields, such as medicine, diagnostics, nanotechnology, and artificial intelligence.