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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【教学内容一】分段盐析法，凝胶过滤法(盐析法概念及原理，分段盐析概念，分子筛效应，柱层析基本技术)
【实验原理】1.盐析法是一种利用蛋白质在高盐溶液中溶解度不同而分离的方法。通过将硫酸铵加入蛋白质溶液中，使蛋白质表面电荷被中和，水化膜被破坏，导致蛋白质在水溶液中的稳定性因素去除而沉淀。2.通过分段改变盐类浓度达到分离目的的方法叫分段盐析法。3.凝胶过滤又称分子筛层析。混合物随流动相流经装有凝胶作为固定相的层析柱时，分子量大的物质因不能进入凝胶网孔而沿凝胶颗粒间的空隙先被洗脱(阻力小,流程短,流速大),分子量小的物质因能进入凝胶网孔而受阻滞,流速缓慢,流程长而后被洗脱,由于流速不同可以把大小不同的分子分开.
【仪器与试剂】1.离心机2.真空泵3.饱和硫酸铵溶液：称取767g固体硫酸铵，加入1000ml水中，加热使之溶解，室温冷却后4℃静置过夜，然后用氨水将pH调至中性。4.固体(NH4)2SO45.SephadexG—256.奈氏试剂7.双缩脲试剂8.pH7.4,0.05MPBS
【步骤】
一分段盐析
1.观看人血清抗胰蛋白酶(α1-AT)的分离及鉴定教学录像2.每组量取20ml血清，倒入一干净烧杯中，缓慢加入饱和硫酸铵20ml，边加边搅拌,放入4℃冰箱30分钟3.从冰箱中取出烧杯，将血清倒入离心管，3000rpm，离心20分钟4.将上清倒入一干净量筒中，记录体积后，倒入干净烧杯中，按17.6g/100ml量取固体硫酸铵，缓慢加入上清中，边加边搅拌。4℃冰箱放置30分钟5.将烧杯中液体倒入抽滤器中，负压抽滤6.刮下滤纸上的粗提蛋白，用4ml缓冲液溶解，准备加样
二凝胶过滤层析
1.凝胶柱准备：(1)连接层析柱(2)夹住出口，加入1/3体积的0.05MpH6.4PBS，灌胶，待凝胶自然沉降约1cm时，打开出口，控制流速，60滴/分(3)层析柱填装完成后，连接下口瓶，调节流速，使入口和出口流速相同。平衡至入口pH值与出口pH值相同(即pH试纸呈色相同)，关闭出口，等待加样2.加样：用吸管吸去胶面以上的缓冲液，立即加入蛋白粗提液(沿管壁缓慢加入)3.打开出口，调流速15滴/分，待蛋白粗提液完全进入胶面，用少量缓冲液清洗管壁。连接下口瓶4.检测：用双缩脲试剂检测洗脱液。待试剂变红，开始收集，直到试剂不变色停止收集。测量并记录收集液的体积，留取1ml样品(标记为G—25样品)，放入一冷冻管中冰箱冻存;剩余的液体收集入一大试管中，作好标记，下次实验用5.洗去铵盐：全速洗脱，用奈氏试剂检测洗脱液，直到橙色消失为止，回收凝胶
【注意事项】1.盐析所用器皿一定要干燥2.盐析时，应将饱和硫酸铵或固体硫酸铵加入到血清中，且边加边搅拌3.层析柱上方要留有少量液体，避免使凝胶暴露于空气中4.凝胶过滤上样时，使样品沿管壁缓缓流下，勿冲破胶面
（信息提供：探生网生物医药）
有机溶剂沉淀，极性有机溶剂降低介点常数使蛋白质凝集沉淀。eg.甲醇、乙醇、丙酮。
磺基水杨酸也可以沉淀蛋白质，但要加酸调节ph，使之低于血清蛋白的pi，使蛋白质带正电荷，与水杨酸酸根结合生成不溶盐而沉淀。
血清蛋白质均带负电荷，但各种蛋白质带负电荷的程度有所差异，以白蛋白为最多。而血清白蛋白等电点为4.7－4.9。所以把ph调到4.7以下就可以了。
血清为去除纤维蛋白原后的无形成分，颜色微黄，透亮。
血清的基本成分是水，水中溶有蛋白质、脂肪、糖、无机盐、维生素等营养成分，也溶有人体代谢产物。血清中有抗体，这是被称作免疫球蛋白的蛋白质。
