Characterization of a polyclonal antibody directed against the amino terminus of the human sodium‐coupled bicarbonate transporter NCBE

Abstract

The SLC4 family of bicarbonate transporters includes the products of ten genes. These transporters play an essential role in intracellular-pH regulation, transepithelial HCO3 −transport, and CO2 transport by the RBC. Because the sequences of SLC4 members are highly conserved, it is important for the study of the SLC4 proteins to develop antibodies that can distinguish different members of the family. NCBE is an electroneutral Na+-coupled HCO3 −transporter that is mainly expressed in central nervous system. Here we characterized the specificity of a new antibody that we generated in a rabbit against a fusion protein consisting of maltose-binding protein (MBP) and the first 135 amino acids at the N-terminus of human NCBE. The two SLC4 proteins most closely related to NCBE are the human Na+-driven Cl-HCO3 exchanger NDCBE and the electroneutral Na/HCO3 cotransporter NBCn1. By western blotting, the antiserum at a dilution of 1:10,000 recognized a purified peptide representing the first 124 N-terminal amino acids (aa) of NCBE, but not comparable peptides for NDCBE (116 aa) or NBCn1 (123 aa). Also by western blotting, the antiserum at a dilution of 1:2000 recognized full-length NCBE expressed in Xenopus oocytes, but not full-length versions of either NDCBE or NBCn1 expressed in oocytes. Thus, the antiserum is specific for NCBE and does not cross react with the two SLC4 members that are most highly homologous to NCBE. (This work is supported by NS18400.)