title: Two-dimensional gel electrophoresis and immunoblot analysis of Neospora caninum tachyzoites.
abstract: Identification of expressed protein profiles and antigenic determination are some of the most challenging aspects of proteomics. Two-dimensional gel electrophoresis (2-DE) combined with immunoblot analysis were employed to study the N. caninum proteome. Protein sample preparation was carried out by first conducting sonication, followed by adding lysis buffer containing 7M urea plus 2M thiourea to the purified tachyzoites in order to complete disruption. A total of 335 differentially expressed protein spots were detected using pH 4-7 IPG strip (7 cm) that were run in a 56 kVh isoelectric focusing (IEF) system. Of the spots analyzed, 64 were identified as antigenic spots on immunoblot profile. Major antigenic spots appeared at 65 kDa (pI 5.2-5.3), 51 kDa (pI 5.5), 38 kDa (pI 5.1), 33 kDa (pI 4.4), 29 kDa (pI 5.6) and 15.5 kDa (pI 5.0) were observed to be significantly distinct compared to the rest of the antigenic spots. The results indicate that combination of 2-DE and immunoblotting methods were thought as very useful tools in defining both proteins and antigens of N. caninum tachyzoites. Additionally, present 2-DE profiles may be valuable in further proteomic approaches and study of the pathogen.
authors.affiliation: Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju 600-701, Korea.
authors.forename: Eung-Goo
authors.initials: EG
authors.lastname: Lee
authors.forename: Jae-Hoon
authors.initials: JH
authors.lastname: Kim
authors.forename: Yong-Seung
authors.initials: YS
authors.lastname: Shin
authors.forename: Gee-Wook
authors.initials: GW
authors.lastname: Shin
authors.forename: Yong-Hwan
authors.initials: YH
authors.lastname: Kim
authors.forename: Gon-Sup
authors.initials: GS
authors.lastname: Kim
authors.forename: Dae-Yong
authors.initials: DY
authors.lastname: Kim
authors.forename: Tae-Sung
authors.initials: TS
authors.lastname: Jung
authors.forename: Myung-Deuk
authors.initials: MD
authors.lastname: Suh
mesh_terms: D000818:Animals; D000917:Antibody Formation; D000953:Antigens, Protozoan / Q000032:analysis* / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000032:analysis; D007091:Image Processing, Computer-Assisted; D015151:Immunoblotting / Q000379:methods; D007525:Isoelectric Focusing; D018688:Neospora / Q000737:chemistry* / Q000276:immunology; D020543:Proteome / Q000032:analysis; D040901:Proteomics; D015800:Protozoan Proteins / Q000032:analysis* / Q000302:isolation & purification
chemical_list: D000953:Antigens, Protozoan; D000939:Epitopes; D020543:Proteome; D015800:Protozoan Proteins