Synthesis and evaluation of fluorescent chemokines labeled at the amino terminal

Abstract

Publisher Summary Chemokines are a rapidly growing family of proinflammatory cytokines that are involved in cell recruitment and activation. This chapter observes the direct binding of chemokines by techniques such as fluorescence activated cell sorting (FACS) analysis and confocal microscopy. Binding with fluorescent ligands has been limited to the commercially available phycoerythrin-IL-8 conjugate, which consists of the addition of a relatively large fluorescent moiety of 240 kDa attached to the 8-kDa protein through its side chains. Such a conjugate is many times larger than the chemokine itself, and because there are so many side chains available for conjugation, the product is likely to be a mixture of structural isomers. Therefore a highly controlled, site-specific reaction is sought to attach fluorophores to the polypeptide chain to ensure that each chain had one fluorophore, always at the same, chosen position in the chain. The chemical reactions involved had to be efficient and, so as not to compromise the biological behavior of the products, had to take place under mild, aqueous conditions.