Alpha-thrombin is a highly specific serine protease generated by proteolytic activation of the zymogen prothrombin (1). During coagulation, thrombin cleaves fibrinogen to form fibrin, leading to the ultimate step in coagulation, the formation of a fibrin clot. Thrombin is also responsible for feedback activation of the procofactors factor V and factor VIII. Thrombin has also been reported to activate factor XIII and platelets, and also functions as a vasoconstrictor protein. The procoagulant activity of thrombin is arrested in two ways: 1) inhibition by either heparin cofactor II or the antithrombin III/heparin complex; or 2) complex formation with thrombomodulin. Formation of the thrombin/thrombomodulin complex results in the inability of thrombin to cleave fibrinogen and activate factors V and VIII, but increases the efficiency of thrombin for activation of the anticoagulant, protein C.
Thrombin is a two chain enzyme composed of an NH2-terminal "A" chain (Mr=6,000) and a COOH-terminal "B" chain (Mr=31,000) which remain covalently associated through a single disulfide bond. Human thrombin is 13 amino acids shorter than the bovine thrombin due to a thrombin cleavage site on the human protein that is not present in the bovine protein.
Thrombin is also utilized for site specific cleavage of fusion proteins expressed in bacteria (9-11). A thrombin sensitive site is incorporated between the recombinant protein of interest and peptides or proteins which facilitate purification and/or expression. The target protein is released from the expressed hybrid by cleavage with thrombin. Thrombin can then be easily removed by affinity chromatography.
Human, bovine and mouse thrombin are prepared from purified prothrombin using a modification of the Lundblad procedure (1) as described by Nesheim et al. (2). Thrombin is supplied in 50% (vol/vol) glycerol/H2O and should be stored at -20oC. Purity is determined by SDS-PAGE analysis and activity is measured in a thrombin specific clotting assay, and compared to standardized NIH thrombin. Thrombin is also available with the active site blocked with either DFP, FPRck, or biotinlyated FPRck.
Cleavage of Fusion ProteinsIn addition to its broad application in coagulation research thrombin can be used for site specific cleavage of fusion proteins. A thrombin sensitive site is incorporated between the recombinant protein of interest and peptides or proteins which facilitate purification and/or expression. The target protein is released from the expressed hybrid by cleavage with thrombin. Thrombin can then be easily removed by affinity chromatography. Lot to lot consistency ensures reproducible results every time. For experiments involving cell cultures, please contact us to discuss custom, low endotoxin lots designated for cell culture use.
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谢谢各位
非常急!
一、分离脂蛋白
血浆是分离纯化脂蛋白的首选标本。从血浆(血清)中可分离得到CM、VLDL、LDL和HDL,再经脱脂除去脂蛋白中的脂质,剩下的部分为载脂蛋白的混合物。这一操作过程是获得载脂蛋白的最佳方法。
二、脱脂
由于各种脂蛋白含脂的种类和质量不同,脱脂剂组成略有差异。目前常用的几种脱脂方法有如下几种。
1.Warnick等法
将纯化的脂蛋白慢慢加入到预冷(-20℃)的丙酮:无水乙醇(1:1,V/V)混合液中,脱脂液的量为样品的20倍,不断搅拌,然后在-20℃冰箱放置过夜,4℃离心沉淀,将沉淀重复脱脂一次,用氮气吹干或真空干燥,脱脂物0℃贮存待用。
2.Scanu等法
将脂蛋白加入到预冷的(-15℃)无水乙醇:无水乙醚(3:2)混合液中,不断搅拌,以12h过夜,在-10℃或4℃离心(2000r/min),去上清留沉淀,再重复脱脂一次。用N2或真空干燥,0℃贮存待用。
盐析 ——用于各种蛋白质酶离纯化;
蛋白质溶液加入量性盐破坏蛋白质胶体稳定性使其析种称盐析用性盐硫酸铵、硫酸钠、氯化钠等各种蛋白质盐析所需盐浓度及pH同故用于混蛋白质组离例用半饱硫酸铵沉淀血清球蛋白饱硫酸铵使血清白蛋白、球蛋白都沉淀盐析沉淀蛋白质经透析除盐仍保证蛋白质性调节蛋白质溶液pH至等电点再用盐析则蛋白质沉淀效更盐析两类第类叫Ks段盐析定PH温度通改变离强度实现用于早期粗提液;第二种叫b段盐析定离强度通改变PH温度实现用于期进步离纯化结晶影响盐析素包括:蛋白质浓度、离强度类型、PH值、温度等针温度条需要强调:低离强度或纯水蛋白质溶解度定范围内随温度增加增加高浓度蛋白质、酶肽类物质溶解度随温度升降般情况蛋白质盐析温度特殊要求室温进行某些温度比较敏酶要求0-4℃进行
使用硫酸铵沉淀蛋白需要注意:硫酸铵含少量重金属离蛋白质巯基敏作用使用前必须用H2S处理:硫酸铵配浓溶液通入H2S饱放置夜用滤纸除重金属离浓缩结晶100℃烘干使用另外高浓度硫酸铵溶液般呈酸性(PH=5.0左右)使用前需要用氨水或硫酸调节至所需PH
机溶剂沉淀 ——用于物、糖及核酸产品离纯化;
机溶剂沉淀机理降低水介电数导致具表面水层物脱水相互聚集析该优点于:1)辨能力比盐析高即蛋白质或其溶剂比较窄机溶剂浓度沉淀;2)沉淀用脱盐滤较容易;3)化制备应用比盐析广泛温机溶剂沉淀蛋白质往往引起变性例酒精消毒灭菌操作要求低温进行机溶剂选择首先能水混溶使用较机溶剂乙醇、甲醇、丙酮二甲基甲酰胺、二甲基亚砜、乙腈2-甲基-2,4戊二醇等
等电点沉淀 ——单独应用较少与其结合使用;
两性电解质净电荷零溶解度低同两性电解质具同等电点基础进行离工业产胰岛素粗提液先调PH8.0除碱性蛋白质再调PH3.0除酸性蛋白质利用等电点除杂蛋白必须解制备物酸碱稳定性盲目使用十危险少蛋白质与金属离结合等电点发偏移故溶液含金属离必须注意调整PH值等电点与盐析、机溶剂沉淀或其沉淀联合使用提高其沉淀能力
重金属盐沉淀 ——用于抢救误服重金属盐毒病;
许机物质包括蛋白内碱性溶液带负电荷能与金属离形沉淀根据机物与间作用机制羧酸、胺及杂环等含氮化合物类铜锌镉;亲羧酸疏含氮化合物类钙镁铅;亲硫氢基化合物类汞银铅蛋白质-金属离复合物重要性质溶解度溶液介电数非敏调整水溶液介电数(加入机溶剂)即沉淀种蛋白沉淀条件pH稍于等电点宜重金属沉淀蛋白质变性若低温条件并控制重金属离浓度用于离制备变性蛋白质临床利用蛋白质能与重金属盐结合种性质抢救误服重金属盐毒病给病口服量蛋白质用催吐剂结合重金属盐呕吐解毒
①在用超滤除白蛋白,IgG中硫酸铵盐时,膜包该如何选择?选择几个?
②如用凝胶过滤来分级纯化血清中65%硫酸铵盐析出的白蛋白和纯化33%硫酸铵盐析出的IgG,其填充介质应选择什么?其洗脱缓冲液应用什么好?
下面有些关于这方面的数据,供参考:
①牛血清白蛋白:分子量:66210;分子形状:椭圆形;分子大小:
40Å*140Å;等电点:4.7;血浆中的含量:52.0g/L。
②IgG:分子量:15300;分子形状:球状;等电点:5.8—7.3;血浆中含量:2.0g/L。
③另外,我从书上看到说:凝胶过滤在分级方法中分辨率为中等,但对脱盐效果优良;流速较低,对分级每周期约≥8小时,对脱盐仅30分钟;适用于大规模纯化的最后步骤,在纯化过程的任何阶段均可进行脱盐处理,尤其适用于两种缓冲液交替时。
期待您的帮助,谢谢您。
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