Two-dimensional gel electrophoresis and immunoblot analysis of Neospora caninum tachyzoites.
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.
Lee
Eung-Goo
EG
Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Jinju 600-701, Korea.
Kim
Jae-Hoon
JH
Shin
Yong-Seung
YS
Shin
Gee-Wook
GW
Kim
Yong-Hwan
YH
Kim
Gon-Sup
GS
Kim
Dae-Yong
DY
Jung
Tae-Sung
TS
Suh
Myung-Deuk
MD
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
D000953:Antigens, Protozoan; D000939:Epitopes; D020543:Proteome; D015800:Protozoan Proteins