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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这样可不可以？
另还有几个问题：1、这样的裂解液裂解线粒体提蛋白可不可以
2、裂解液加多少剂量
3、12000g离心10min和30分钟有没有区别
(1)1×107细胞重悬,其来源可为新鲜样品或贮存于-80℃1mol冰冻清洗缓冲液中
的无DMSO的样品,冰冻缓冲液包括10mMHEPES(pH7.5),1.5mMMgCl2,10mMKCl,
1mM二硫苏糖醇(DTT),然后离心(16000rpm、4℃、1min)。
(2)重悬细胞团于100ul冰冻裂解缓冲液,其包含10mMTris-HCl(pH7.5),1mMMgCl2,1mMEGTA,0.1mM苯甲基磺酰氟(DMSF),5mMB2巯基乙醇,0.5%CHAPs(Pierce)和10%甘油。
(3)冰育30分,然后在超速离心机中离心30分钟。
(4)去上清,液氮速冻沉淀,裂解细胞于-80℃保存。
二、扩增
(1)在微量管中冻干0.1ug荧光标记的cx反转引物,蜡封(5′CCCTTACCCTTACCC
TTACCCTTA3′)。
(2)于蜡界之上加50ulTRAP反应物,包括Tris-Cl(pH8.3)20mM,1.5mMMgCl2,
63mMKCl,0.005%,TWeen20,1mMEGTA,50mM三磷酸脱氧核苷,0.1ug荧光标记
TS前导引物(5′AATCCGTCGAGCAGAGTT3′),1ugT4基因32蛋白,牛血清白蛋白0.1mg/ml,2UTaqNDA聚合酶(Takara,shyzou)和5ugCHAPs细胞提取液。荧光标记TS引物可由市场购得。扩增前5ug提取物与1ugRNA酶37℃水浴20分钟,为了端粒酶活性的标准化,运用体内端粒酶活性标准(TTAS):TS重叠引物和CX重叠引物(TS:5′AATCCGTCGAGCAGAGTTGTGAATGAGGCCTTC3′及CX:5′CCCTTACCCTTACCCTTACCCTTATAGGCGCTCAATGTA3′)TS(18bp)和CX(24bp)序列为普通型,下划线标出的为成肌素序列(每个15bp)。成肌素CDNA通过这些引物扩
增产生了150bp的产物,它可用与ITAS扩增端粒酶梯度相同的TS引物和CX引物进行
再扩增。每一项测定用25attogram以显现I-TAS,它并不干扰TRAP测量。
(3)置于22℃10分钟,作为TS引物由端粒酶介导的延伸。90℃热灭活反应物90秒。
(4)PCR扩增,条件94℃30S,50℃30S,72℃1.5min;共27循环。
三、分析
(1)制备8%的变性胶(longRanger,ATBiochem)含6M尿素。
(2)加电泳缓冲液(1×TBE)于电泳槽。
(3)上样缓冲液与产物各1ul混合后上样,上样缓冲液含90%甲酰胺和10%蓝葡聚
糖(Bluedertian)。
(4)95℃预发性4分钟,速冷,上样,运用100bp,150bp,200bp大小荧光标记物。
(5)测序
(6)测序结果由FiagmuntManager程序自动分析,每一荧光峰根据大小、高度、面积定
量。因为过量扩增的产物会产生不可信的面积值,一部分PCR产物应在蒸馏水稀释后再分析
裂解液裂解是一种比较温和的红细胞去除方法,主要用于经酶消化分散的组织细胞的分离纯化，淋巴细胞的分离纯化以及组织细胞蛋白与核酸提取等实验中红细胞的去除。经红细胞裂解液裂解得到的组织细胞中不含红细胞，可进一步用于原代培养、细胞融合、流式细胞分析、核酸与蛋白的分离和提取等。
用SDS裂解，RNA酶降解，
在过柱，后洗脱
不知单纯使用冰浴中超声裂解够了吗？还是说要选用裂解液呢？最主要的就是我不知道该怎么赔这个裂解液，使其COX活性不产生影响
