Two-dimensional gel electrophoresis of Helicobacter pylori for proteomic analysis.
- Author:
Tae Sung JUNG
1
;
Seung Chul KANG
;
Yeo Jeong CHOI
;
Beong Sam JEON
;
Jeong Won PARK
;
Sun Ae JUNG
;
Jae Young SONG
;
Sang Haeng CHOI
;
Seong Gyu PARK
;
Mi Young CHOE
;
Byung Sang LEE
;
Eun Young BYUN
;
Seung Chul BAIK
;
Woo Kon LEE
;
Myung Je CHO
;
Hee Sang YOUN
;
Gyung Hyuck KO
;
Kwang Ho RHEE
Author Information
1. Department of Microbiology, Gyeongsang National University College of Medicine, Chinju, Kyung-Nam, 660-751, South Korea. khrhee@gshp.gsnu.ac.kr
- Publication Type:Original Article
- MeSH:
Electrophoresis;
Electrophoresis, Gel, Two-Dimensional*;
Helicobacter pylori*;
Helicobacter*;
Hydrogen-Ion Concentration;
Molecular Weight;
Protein Isoforms;
Proteomics;
Proton-Motive Force;
Silver Staining;
Sodium;
Urea
- From:Journal of the Korean Society for Microbiology
2000;35(2):97-108
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
Two-dimensional gel electrophoresis (2-DE) is an essential tool of proteomics to analyse the entire set of proteins of an organism and its variation between organisms. Helicobacter pylori was tried to identify differences between strains. As the first step, whole H. pylori was lysed using high concentration urea contained lysis buffer (9.5 M Urea, 4% CHAPS, 35 mM Tris, 65 mM DTT, 0.01% SDS and 0.5% Ampholite (Bio-Rad, pH 3-10)). The extract (10 mug) was rehydrated to commercially available immobilised pH gradient (IPG) strips, then the proteins were separated according to their charges as the first dimensional separation. The IPG strips were placed on Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to separate according to molecular mass of the proteins as the second dimension. The separated protein spots were visualised by silver staining in order to compare different expression of proteins between strains. Approximately 120 spots were identified in each mini-protein electrophoresised gel, furthermore about 65 to 75 spots were regarded as identical proteins in terms of pI value and molecular weight between strains used. In addition, distinct differences were found between strains, such as 219-1, Y7 and Y14, CH150. Two representative strains were examined using strips which had pH range from 4 to 7. This strips showed a number of isoforms which were considered large spots on pH range 3-10. Furthermore, the rest of spots on pH 4-7 IPG strips appeared very distinctive compared to broad range IPG strips. 2-DE seems to be an excellent tool for analysing and identifying variations between H. pylori strains.
