J Vet Sci.  2004 Jun;5(2):139-145.

Two-dimensional gel electrophoresis and immunoblot analysis of Neospora caninum tachyzoites

• Lee EG
• Kim JH
• Shin YS
• Shin GW
• Kim YH
• Kim GS
• Kim DY
• Jung TS
• Suh MD

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.

Keyword

Neospora caninum; two-dimensional gel electrophoresis(2-DE); immunoblot

MeSH Terms

Animals
Antibody Formation
Antigens, Protozoan/*analysis/isolation&purification
Electrophoresis, Gel, Two-Dimensional/methods
Electrophoresis, Polyacrylamide Gel
Epitopes/analysis
Image Processing, Computer-Assisted
Immunoblotting/methods
Isoelectric Focusing
Neospora/*chemistry/immunology
Proteome/analysis
Proteomics
Protozoan Proteins/*analysis/isolation&purification