ZERO-LENGTH CROSSLINKING PROCEDURE WITH THE USE OF ACTIVE ESTERS
ZERO-LENGTH CROSSLINKING PROCEDURE WITH THE USE OF ACTIVE ESTERS
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DOI：
10.1016/0003-2697(90)90267-d
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发表时间：
1990-02-15
影响因子：
2.9
通讯作者：
GERGELY, J
中科院分区：
文献类型：
--
作者：
GRABAREK, Z;GERGELY, J
开发了一种用于研究蛋白质-蛋白质复合物的双sep零长度交联方法。在n -羟基琥珀酰亚胺的存在下，将配合物的一个组分与1-乙基-3-（3-二甲氨基丙基）碳二亚胺（EDC）短暂孵育，导致一些蛋白质羧基转化为琥珀酰亚胺酯。加入-就停止了反应。然后加入巯基乙醇和其他相互作用的蛋白质。交联是由赖氨酸取代产生的。在1至2小时的潜伏期内，这些蛋白质的-氨基为琥珀酰基部分。与一步零长度交联相比，这种方法的优点是只有一个组分的配合物暴露在交联剂中，这消除了多组分配合物的几个蛋白质之间形成交联所引起的并发症。此外，即使存在试剂，如二硫苏糖醇和EDTA，也可以形成交联，这些试剂会干扰与EDC的直接交联。
A two-sep zero-length crosslinking procedure for studying protein-protein complexes has been developed. One component of a complex is briefly incubated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of N-hydroxysuccinimide resulting in the conversion of some of the protein carboxyls into succinimidyl esters. The reaction is stopped by addition of .beta.-mercaptoethanol and other interacting proteins are then added. Crosslinking arises from substitution of lysine .epsilon.-amino grops of these proteins for the succinimidyl moieties during a 1- to 2-h incubation period. The advantage of this method versus one-step zero-length crosslinking is that only one component of the complex is exposed to the crosslinker, which eliminates complications arising from the formation of crosslinks among several proteins of a multicomponent complex. Furthermore, crosslinks can be formed even in the presence of reagents, such as dithiothreitol and EDTA, that would interfere with direct crosslinking with EDC.