我们知道Cys存在与许多蛋白中，且其中的－SH常作为活性中心发挥作用，或者，两个－SH连接成二硫键，是维持蛋白质高级构象重要桥梁。 －SH具有很强的还原性，溶液被溶液中的溶解氧或其它氧化物氧化，从使蛋白失去活性。 所以，在蛋白纯化和保存时，在溶液中加入一定量的DTT，以免蛋白－SH被氧化而失活。
但这里就有一个矛盾，DTT该加多少量，以使－SH保持还原状态，而使二硫键免遭还原？
除了上述作用，DTT还有哪些方面可以起到作用？
我的知识面非常有限，请各位战友多多指教。
也可以谈谈其它相关的，比如说重金属离子会与－SH发生反应，生成硫醇盐，因此可以在溶液重添加适量EDTA，螯合掉金属离子。
According to my experience and experiment data, one cys should be added 5 times DTT.
Usualy, we add ten times DTT, and that really work to keep proteins stable
我也曾听一高人说起过，以摩尔数计算，5-10倍的量来保护－SH。
如果蛋白报存buffer中含有15％左右的甘油，对蛋白具有很好的稳定作用。
呵呵，补充一点：如果dtt和蛋白摩尔比例比例增大到1：100，DTT就能破坏2硫键了，这时对应的浓度一般在10mM左右，这个一般在做还原肽图的时候用到。
不过有些分子内的二硫键由于空间位阻的作用，还原剂的量增加到很大也不能破坏。这时可能要配合尿素等变性剂才能彻底打断二硫键（如包涵体变性）
DTT在离子状态才有作用，在pH7-9.5的时候作用最强，pH降到3以下几无电离，因此可以通过降低pH至3以下来终止DTT作
现在才知道DTT作用还受到pH的影响。
提到尿素变性，要是蛋白存在分子间或分子内二硫键，即使具有高浓度的尿素或盐酸胍，而没有DTT等还原剂，那是否也不能使蛋白完全变性（断裂二硫键）？
我记得有篇“从纯化到星辰”文章中提到“万金油”配方是这样的:
50mM Tris pH 7.9
0.5mM EDTA 中华护理杂志>优酷盛典
50mM NaCl
5% glycerol
笔者用该配方复性某个蛋白，比某个试剂盒中十几种buffer的效果都好。
数据库系统
DTT断裂二硫键需要什么的条件？蛋白的二硫键断裂后，形态、功能等发生怎样的改变？
自然界大事件我不是专门搞生化的。只是知道点皮毛而已，就我了解的而言，western blot要得以进行，特别是抗体要和目的蛋白结合，一方面要使打开蛋白质的SS键，因为如果SS键不打开，很有可能抗体不能很好的识别特定的位点，就不能结合上去；另一方面还要用SDS-PAGE消除蛋白质分子内的各种非共价键，使蛋白质所带电荷和其分子量成正比，这样才能使蛋白质分离出来。
了解的不是很确切，希望大家指正
DTT在电泳中还有一个作用。过量DTT能打开多聚体内的二硫键，使呈各个单体状态，而在loading中去除DTT后，多聚体仍然保持原状态。因此两者对比可以分辨该蛋白是否含有多聚体形式。
结合我的实验，我做的是有关纤维素酶基因的克隆与表达。应用的是家蚕杆状病毒表达系统。 携带有纤维素酶基因片段的重组病毒构建好以后，将转染细胞所获得的达到一定滴度的重组病毒从皮下注射到家蚕体内。
家蚕血淋巴中是否能表达纤维素酶？这是我目前所研究的重点。难点以及疑惑：家蚕血淋巴在空气中容易被氧化，虽然低温下可保持短时间内不被黑化（即氧化），但测酶活的时候需要经过50度反应30分钟这一步，所以我第一次做的时候由于没添加巯基乙醇或者DTT，所以溶液变黑了。
为什么当时没添加呢？我是出于这样的顾忌：毕竟巯基乙醇或者DTT是强还原剂，这个添加的量掌握不好的话，势必会破坏纤维素酶的天然构象（假设纤维素酶能够表达在家蚕血淋巴），那么这样还能测出酶活吗？换句话说，这样测出的结果还准吗？
今天晚上准备做第二批了，还没想好添加多少量呢，思考ING……也希望各位战友能提供点思路，不甚感激~！
不过今天是周末呀。估计不知要等多少时间要能求得答复呢？o(∩_∩)o...
按照上面版主说的5－10倍于二硫键的量加DTT，做几个梯度看看效果。
关注中，好帖
那我如何确定2硫键的量呢？
DTT在反应液中的终浓度好像是0.5-1mM/L来着。
血清中的组分非常复杂，所以很难用给个具体的值。正如上一层战友所说，用梯度实验看效果。一般的经验值是1mM，而血清中蛋白浓度是比较高的，特别是蚕血中还原物质含量特别高，因此建议往上调
请教：如果多聚体不仅以二硫键维持其相互作用，还以疏水键维持其作用的话，以DTT还能使它完全解聚么？在loading里加入DTT和不加DTT效果会不会差不多？
疏水键是非共价键形式的相互作用，其作用力小于二硫键。
如果是疏水作用，在SDS存在，煮沸的变性条件，完全可以使其解离。DTT只是针对二硫键作用，所以如果知道目的蛋白中二硫键含量高时，要调整loading配方，增加DTT含量。
Dithiothreitol (DTT) is the common name for a small-molecule redox reagent known as Cleland's reagent. DTT's formula is C4H10O2S2 and the molecular structure of its reduced form is shown at the right; its oxidized form is a disulfide-bonded 6-membered ring (shown below). Its name derives from the four-carbon sugar, threose. DTT has an epimeric ('sister') compound, dithioerythritol (DTE).
DTT is an unusually strong reducing agent, owing to its high conformational propensity to form a six-membered ring with an internal disulfide bond. It has a redox potential of -0.33 V at pH 7. The reduction of a typical disulfide bond proceeds by two sequential thiol-disulfide exchange reactions and is illustrated below. The intermediate mixed-disulfide state is unstable (i.e., poorly populated) because the second thiol of DTT has a high propensity to close the ring, forming oxidized DTT and leaving behind a reduced disulfide
bond. The reducing power of DTT is limited to pH values above ~7, since only the negatively charged thiolate form -S– is reactive (the protonated thiol form -SH is not); the pKa of thiol groups is typically ~8.3.
Applications
A common use of DTT is as a reducing or "deprotecting" agent for thiolated DNA. The terminal sulfur atoms of thiolated DNA have a tendency to form dimers in solution, especially in the presence of oxygen. Dimerization greatly lowers the efficiency of subsequent coupling reactions such as DNA immobilization on gold in biosensors. Typically DTT is mixed with a DNA solution and allowed to react, and then is removed by filtration (for the solid catalyst) or by chromatography (for the liquid form). The DTT removal procedure is often called "desalting."
DTT is frequently used to reduce the disulfide bonds of proteins and, more generally, to prevent intramolecular and intermolecular disulfide bonds from forming between cysteine residues of proteins. However, even DTT cannot reduce buried (solvent-inaccessible) dis
ulfide bonds, so reduction of disulfide bonds is sometimes carried out under denaturing conditions (e.g., at high temperatures, or in the presence of a strong denaturant such as 6 M guanidinium hydrochloride, 8 M urea, or 1% Sodium dodecylsulfate). Conversely, the solvent exposure of different disulfide bonds can be assayed by their rate of reduction in the presence of DTT.
雪地里的红棉袄
DTT can also be used as an oxidizing agent. Its principal advantage is that effectively no mixed-disulfide species are populated, in contrast to other agents such as glutathione. In very rare cases, a DTT adduct may be formed, i.e., the two sulfur atoms of DTT may form disulfide bonds to different sulfur atoms; in such cases, DTT cannot cyclize since it has no remaining free thiols.
Properties
Due to air oxidation, DTT is a relatively unstable compound whose useful life can be extended by refrigeration and handling in an inert atmosphere. Since protonated sulfurs have lowered nucleophilicities, DTT becomes less potent as the pH lowers. Tris(2-carbox
yethyl)phosphine HCl (TCEP hydrochloride) is an alternative which is more stable and works even at low pH
豫公网安备41160202000603
站长QQ:729038198
关于我们
投诉建议