S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Food and Animal Biotechnology (식품·동물생명공학부) Theses (Master's Degree_식품·동물생명공학부)
대장균에서 발현된 exoinulinase와 endoinulinase의 이차원적 전기영동과 MALDI-TOF MS를 이용한 동정
- Issue Date
- 서울대학교 대학원
- two-dimensional electrophoresis; Two-dimensional electrophoresis; isoelectric focusing; Isoelectric focusing; image analysis; Image analysis; MALDI-TOF MS; Maldi-tof ms; peptide fingerprinting; Peptide fingerprinting
- 학위논문(석사)--서울대학교 대학원 :농생명공학부,2003.
- Protein identification in proteomic analysis requires many sequential steps
including sample preparation, IEF, two dimensional electrophoresis (2-DE),
image analysis, in-gel digestion with trypsin, matrix assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and
searches of molecular weight in peptide-mass databases. Among these steps,
well resolved 2-DE gels are required for successful proteomic analysis.
Resolution in 2-DE depends on many proceeding steps including sample
preparation, isoelectric focusing (IEF) and SDS-PAGE. However, due to
variations in these steps 2-DE does not always result in reproducible resolutions.
Optimization of these procedures is a foremost important issue in successful
In this thesis, the following procedures were optimized using bacterial
proteins from E. coli BL21; i) sample preparation, ii) optimized Vhr in IEF, iii)
12.5% SDS-PAGE with fresh running buffer and iv) silver staining. These
optimized procedures produced a well-resolved 2-DE gel. Image analysis by
ImageMaster program followed by the in-gel digestion with trypsin identified
unique peptide peaks in MALDI-TOF MS.
These optimized conditions were employed to identify an exo- and an endoinulinase
protein which were expressed in E. coli BL21 with the introduced expression vector. Those two proteins were detected in 2-DE. Further
identifications using peptide fingerprinting in MALDI-TOF analysis clearly
identified these two foreign proteins along with other vector-encoded proteins.
These results support that the optimized procedures in this thesis are well
suitable for analyzing bacterial proteins.
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