title: Validation of endothelin B receptor antibodies reveals two distinct receptor-related bands on Western blot.
abstract: Antibodies are important tools for the study of protein expression but are often used without full validation. In this study, we used Western blots to characterize antibodies targeted to the N or C terminal (NT or CT, respectively) and the second or third intracellular loop (IL2 or IL3, respectively) of the endothelin B receptor (ETB). The IL2-targeted antibody accurately detected endogenous ETB expression in rat brain and cultured rat astrocytes by labeling a 50-kDa band, the expected weight of full-length ETB. However, this antibody failed to detect transfected ETB in HEK293 cultures. In contrast, the NT-targeted antibody accurately detected endogenous ETB in rat astrocyte cultures and transfected ETB in HEK293 cultures by labeling a 37-kDa band but failed to detect endogenous ETB in rat brain. Bands detected by the CT- or IL3-targeted antibody were found to be unrelated to ETB. Our findings show that functional ETB can be detected at 50 or 37kDa on Western blot, with drastic differences in antibody affinity for these bands. The 37-kDa band likely reflects ETB processing, which appears to be dependent on cell type and/or culture condition.
authors.affiliation: Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham & Women's Hospital, Boston, MA 02115, USA.
authors.forename: Travis P
authors.initials: TP
authors.lastname: Barr
authors.affiliation: Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham & Women's Hospital, Boston, MA 02115, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kornberg
authors.affiliation: Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham & Women's Hospital, Boston, MA 02115, USA.
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Montmayeur
authors.affiliation: Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham & Women's Hospital, Boston, MA 02115, USA.
authors.forename: Melinda
authors.initials: M
authors.lastname: Long
authors.affiliation: Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham & Women's Hospital, Boston, MA 02115, USA.
authors.forename: Stephen
authors.initials: S
authors.lastname: Reichheld
authors.affiliation: Pain Research Center, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham & Women's Hospital, Boston, MA 02115, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA. Electronic address: gstrichz@zeus.bwh.harvard.edu.
authors.forename: Gary R
authors.initials: GR
authors.lastname: Strichartz
mesh_terms: D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D001253:Astrocytes / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods; D001921:Brain / Q000378:metabolism; D002478:Cells, Cultured; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D051381:Rats; D044023:Receptor, Endothelin B / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism
chemical_list: D000939:Epitopes; D044023:Receptor, Endothelin B; D011994:Recombinant Proteins
keywords: Antibody; Endothelin B receptor; Immunoblot; Western