Fractionation of polyclonal antibodies to fragments of a neuroreceptor using three increasingly chaotropic solvents

We have developed specific antibodies against fragments of anaplastic lymph oma kinase (ALK) in order to develop tools for characterizing the expressio n and biological function of this orphan receptor. The first fragment consi sted of residues 280 to 480 of the murine extracellular domain, was express ed in Escherichia coli (E. coli), purified in the presence of urea from the pellet of mechanically lysed cells and injected into rabbits as an unfolde d protein in urea. The second fragment consisted of residues 1519 to 1619 o f the murine sequence, corresponding to the C-terminal side of the kinase d omain. It was expressed in E. coli as a soluble glutathione-S-transferase f usion protein, purified from the supernatant of broken cells and injected i nto rabbits as a folded protein. Both antisera were purified using antigen affinity chromatography, with the polyclonal antibodies eluted stepwise usi ng three different buffers, 0.1 M glycine, pH 2.9, followed by 7 M urea, pH 4, followed by 6 M guanidine-HCl (GdnHCl), pH 4. Antisera prepared against either antigen contained antibodies that eluted in each of the three pools , indicating that solvents more chaotropic than acid were required to elute antibody populations that were tightly bound to the antigen column. All th ree antibody pools were reactive towards their respective antigens upon Wes tern blot analysis. Purified polyclonal antibodies (pAbs) to both fragments also recognized the full-length protein expressed in Chinese hamster ovary cells. In every case, the pAbs eluting in GdnHCl were the most sensitive f or detecting full-length ALK. (C) 1999 Elsevier Science B.V. All rights res erved.