title: A sandwich ELISA for the conformation-specific quantification of the activated form of human Bax.
abstract: Bcl-2 family proteins are critical regulators of mitochondrial outer membrane permeabilization (MOMP), which represents the point of no return of apoptotic cell death. The exposure of the Bax N-terminus at the mitochondria reflects Bax activation; and this activated configuration of the Bax protein is associated with MOMP. N-terminal exposure can be detected using specific monoclonal and/or polyclonal antibodies, and the onset of activated Bax has extensively been used as an early marker of apoptosis. The protocols of immunoprecipitation and/or immunocytochemistry commonly used to detect activated Bax are long and tedious, and allow semiquantification of the antigen at best. The sandwich ELISA protocol we developed has a 5 ng/mL detection limit and is highly specific for the activated conformation of Bax. This ELISA allows a rapid quantification of activated human Bax in whole cells and isolated mitochondria protein extracts. These properties grant this assay the potential to further clarify the prognostic and diagnostic value of activated Bax in disorders associated with deregulated apoptotic pathways such as degenerative diseases or cancer.
authors.affiliation: Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Care and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Oscar
authors.initials: O
authors.lastname: Teijido
authors.affiliation: Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, NY 10065, USA.
authors.forename: Yogesh Tengarai
authors.initials: YT
authors.lastname: Ganesan
authors.affiliation: Department of Chemistry, California State University, Fresno, CA 93740, USA.
authors.forename: Raul
authors.initials: R
authors.lastname: Llanos
authors.affiliation: Department of Chemistry, California State University, Fresno, CA 93740, USA.
authors.forename: Ashley
authors.initials: A
authors.lastname: Peton
authors.affiliation: Department of Chemistry, California State University, Fresno, CA 93740, USA.
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Urtecho
authors.affiliation: Department of Basic Sciences, New York University, College of Dentistry, New York, NY 10010, USA.
authors.forename: Adauri
authors.initials: A
authors.lastname: Soprani
authors.affiliation: Department of Basic Sciences, New York University, College of Dentistry, New York, NY 10010, USA.
authors.forename: Aimee
authors.initials: A
authors.lastname: Villamayor
authors.affiliation: Merck Serono S.A. Geneva Research Center, Geneva, Switzerland.
authors.forename: Bruno
authors.initials: B
authors.lastname: Antonsson
authors.affiliation: CNRS, Université de Bordeaux, UMR5095, 1 Rue Camille Saint-Saëns, 33077 Bordeaux, France.
authors.forename: Stéphen
authors.initials: S
authors.lastname: Manon
authors.affiliation: Department of Chemistry, California State University, Fresno, CA 93740, USA. Electronic address: ldejean@csufresno.edu.
authors.forename: Laurent
authors.initials: L
authors.lastname: Dejean
mesh_terms: D017209:Apoptosis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D051336:Mitochondrial Membranes / Q000737:chemistry; D010539:Permeability; D011487:Protein Conformation; D051028:bcl-2-Associated X Protein / Q000032:analysis*
chemical_list: D051028:bcl-2-Associated X Protein
keywords: Apoptosis; Bax; Conformation specificity; ELISA; Mitochondria