分析化学
分析化學
분석화학
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY
2009年
7期
1019-1024
,共6页
牙鲆肝%蛋白质组学%差速离心%镉污染%蛋白指示物
牙鲆肝%蛋白質組學%差速離心%鎘汙染%蛋白指示物
아평간%단백질조학%차속리심%력오염%단백지시물
Paralichthys olivaceus liver%proteomics%differential centrifugation%cadmium contamination%protein marker
人工构建镉盐污染源,选用差速离心结合双向凝胶电泳(2D-PAGE)法,高效提取、分离和筛选牙鲆(Paralichthys olivaceus, PO)受镉盐胁迫后的肝脏全蛋白和差异蛋白质.实验结果表明: 选用直接裂解法提取牙鲆肝(PO liver POL)全蛋白质且用2D-PAGE分离,可获得约800个蛋白斑点,其中镉盐诱导了11个差异蛋白斑点.以相对离心力为1000×g、12000×g和100000 ×g 的差速离心法,分别制备了3种沉淀蛋白和1种胞浆蛋白,称为POL组分Ⅰ、POL组分Ⅱ、POL组分Ⅲ和POL组分Ⅳ(胞浆蛋白),蛋白斑点数目分别为380、550、500和850个,总计2280个,明显高于直接裂解法.比较分析法发现,差速离心结合2D-PAGE分离技术可获得牙鲆肝脏受镉盐胁迫后表达的54个差异蛋白质,并适合于用肽质量指纹(peptide mass fingerprint,PMF)图谱技术鉴定.本实验所建立的差速离心结合蛋白质组学技术可高效提取、分离和鉴定组织全蛋白或差异蛋白,并能有效地筛选出蛋白指示物.
人工構建鎘鹽汙染源,選用差速離心結閤雙嚮凝膠電泳(2D-PAGE)法,高效提取、分離和篩選牙鲆(Paralichthys olivaceus, PO)受鎘鹽脅迫後的肝髒全蛋白和差異蛋白質.實驗結果錶明: 選用直接裂解法提取牙鲆肝(PO liver POL)全蛋白質且用2D-PAGE分離,可穫得約800箇蛋白斑點,其中鎘鹽誘導瞭11箇差異蛋白斑點.以相對離心力為1000×g、12000×g和100000 ×g 的差速離心法,分彆製備瞭3種沉澱蛋白和1種胞漿蛋白,稱為POL組分Ⅰ、POL組分Ⅱ、POL組分Ⅲ和POL組分Ⅳ(胞漿蛋白),蛋白斑點數目分彆為380、550、500和850箇,總計2280箇,明顯高于直接裂解法.比較分析法髮現,差速離心結閤2D-PAGE分離技術可穫得牙鲆肝髒受鎘鹽脅迫後錶達的54箇差異蛋白質,併適閤于用肽質量指紋(peptide mass fingerprint,PMF)圖譜技術鑒定.本實驗所建立的差速離心結閤蛋白質組學技術可高效提取、分離和鑒定組織全蛋白或差異蛋白,併能有效地篩選齣蛋白指示物.
인공구건력염오염원,선용차속리심결합쌍향응효전영(2D-PAGE)법,고효제취、분리화사선아평(Paralichthys olivaceus, PO)수력염협박후적간장전단백화차이단백질.실험결과표명: 선용직접렬해법제취아평간(PO liver POL)전단백질차용2D-PAGE분리,가획득약800개단백반점,기중력염유도료11개차이단백반점.이상대리심력위1000×g、12000×g화100000 ×g 적차속리심법,분별제비료3충침정단백화1충포장단백,칭위POL조분Ⅰ、POL조분Ⅱ、POL조분Ⅲ화POL조분Ⅳ(포장단백),단백반점수목분별위380、550、500화850개,총계2280개,명현고우직접렬해법.비교분석법발현,차속리심결합2D-PAGE분리기술가획득아평간장수력염협박후표체적54개차이단백질,병괄합우용태질량지문(peptide mass fingerprint,PMF)도보기술감정.본실험소건립적차속리심결합단백질조학기술가고효제취、분리화감정조직전단백혹차이단백,병능유효지사선출단백지시물.
Both total proteome and differential proteins were effectively extracted, separated and selected by a combined approach of both differential centrifugation and 2D-PAGE in liver of Paralichtys olivaceus( POL) under the stress of cadmium chloride as an artificial pollution source. Approximately 800 spots for extraction of whole protein separated with 2D-PAGE were obtained by direct lysis in the POL. In addition, approximately 11 differential proteins in POL were also obtained under the stress of cadmium chloride. The differential centrifugal were used to prepare three sedimentation and a plasmolysis proteins, called POL component Ⅰ, POL component Ⅱ, POL component Ⅲ, and POL component Ⅳ(plasmolysis protein), respectively. Total protein spots for each gel were calculated to have 380, 550, 500, and 850, respectively, approximately 2280 spots in sum, while total spots are much higher than those by direct lysis approach. Using the comparison method, approximately 54 differential proteins in POL were obtained by a combined technology of both differential and two dimensional polyacylaminde gel electrophoresis(2D-PAGE) methods under the stress of cadmium chloride. In addition, these differential proteins can be further identified by peptide mass fingerprint(PMF). Here, these combined techniques can be effectively used to extract, separate and identify the whole proteins and the differential proteins including protein markers in the biological tissue.
