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zhoulin5861

新虫 (初入文坛)


[交流] 【求助】关于EDC和NHS的合成

想要把-COOH和-NH2连在一起,打算使用EDC和NHS做交联剂,是在水溶液中进行的,想问一下加入顺序以及对溶剂的量的要求,谢谢
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NHS-EDC使用简介

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特战英豪

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小木虫: 金币+0.5, 给个红包,谢谢回帖
Protocol
1.  Dissolve the protein to be activated in 0.05 M MES, 0.5 M NaCl, pH 6.0 (reaction buffer),
at a concentration of 1 mg/ml.
2.  Add to the solution in step 1 a quantity of EDC and sulfo-NHS (Thermo Fisher) to obtain
a concentration of 2 mM EDC and 5 mM sulfo-NHS. To aid in aliquoting the correct
amount of these reagents, they may be quickly dissolved in the reaction buffer at a higher
concentration, and then a volume immediately pipetted into the protein solution to obtain
the proper molar quantities.
3. Mix and react for 15 minutes at room temperature.
4.  Add 2-mercaptoethanol to the reaction solution to obtain a final concentration of 20 mM.
Mix and incubate for 10 minutes at room temperature. Note: If the protein being activated is sensitive to this level of 2-mercaptoethanol, instead of quenching the reaction
chemically, the activation may be terminated by desalting (step 5).
5.  If the reaction was quenched by the addition of 2-mercaptoethanol, the activated protein may be added directly to a second protein or other amine-containing molecule for
conjugation. Alternatively, or if no 2-mercaptoethanol was added, the activated protein
may be purified from reaction by-products by gel filtration using a desalting resin. The
desalting operation should be done rapidly to minimize hydrolysis and recover as much
active ester functionality as possible. The use of centrifugal spin columns of some sort
may afford the greatest speed in purification (Thermo Fisher). After purification, add the
activated protein to the second molecule for conjugation. The second protein or other
amine-containing molecule should be dissolved in 0.1 M sodium phosphate, pH 7.5.
This will bring the pH of the coupling medium above pH 7.0 to initiate the active ester
reaction.
6. React for at least 2 hours at room temperature.
7. Remove excess reactants by gel filtration or dialysis.
13楼2014-07-05 21:26:23
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会游泳的鱼

金虫 (小有名气)


引用回帖:
Originally posted by zhoulin5861 at 2011-03-17 10:24:21:
想要把-COOH和-NH2连在一起,打算使用EDC和NHS做交联剂,是在水溶液中进行的,想问一下加入顺序以及对溶剂的量的要求,谢谢

你的问题解决了吗,我也有这样的困惑,不知道EDC NHS的用量,很苦恼啊
2楼2011-03-21 20:10:17
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wuyuhan1988

金虫 (初入文坛)



小木虫(金币+0.5):给个红包,谢谢回帖
引用回帖:
Originally posted by zhoulin5861 at 2011-03-17 10:24:21:
想要把-COOH和-NH2连在一起,打算使用EDC和NHS做交联剂,是在水溶液中进行的,想问一下加入顺序以及对溶剂的量的要求,谢谢

我也困惑...期待解答
3楼2011-04-19 10:16:20
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tfyeszjwl

至尊木虫 (职业作家)



小木虫(金币+0.5):给个红包,谢谢回帖
应该加料顺序没太大影响吧
4楼2011-04-19 19:51:38
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