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[Bio101] Labeling Protein with Thiol-reactive Probes
[Bio101] 用thiol活性探针标记蛋白   

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Abstract

This protocol is used to label protein or peptide with a maleinmide or iodoacetamide conjugated fluorescent probe through the free cysteine.

Materials and Reagents

  1. Thiol-reactive Maleimide probes (Life Technologies, Invitrogen™)
    Note: A large collection of probes are available from Invitrogen.
  2. Tris-(2-carboxyethyl)phosphine (TCEP) (Life Technologies, Invitrogen™, catalog number: T2556 )
  3. Dithiothreitol (DTT) (Life Technologies, Molecular Probes®, catalog number: D1532 )
  4. DMSO
  5. Dissolving buffer (see Recipes)

Equipment

  1. Aluminum foil
  2. Sephadex G-25 column

Procedure

  1. Dissolve protein at 50-100 μM in dissolving buffer containing 1 mM TCEP at room temperature to keep reactive cysteine reduced.
    Note: It is not necessary to remove excess TCEP during conjugation with iodoacetamides or maleimides. TCEP can be substituted with DTT. If DTT is used, then dialysis is required to remove the excess DTT prior to introducing the reactive dye.
  2. A 1-10 mM stock solution of thiol-reactive probe is prepared in DMSO immediately prior to use. Protect stock solution from light by wrapping containers in aluminum foil.
  3. Mix thiol-reactive probe and protein as approximately 10:1 molar ratio and allow the reaction to proceed preferably in dark for 2 h at room temperature or overnight at 4 °C.
    Note: Add the probe into protein solution dropwise and it is stirring.
  4. Upon completion of the reaction, the conjugate is separated from excess dye on a gel filtration column (e.g. a Sephadex G-25 column) or by extensive dialysis at 4 °C in an appropriate buffer.

Recipes

  1. Dissolving buffer (pH 7.0-7.5)
    10-100 mM phosphate
    Tris
    HEPES

References

  1. Hermanson, G., (1996). Bioconjugate Techniques, Academic Press.

简介

该方案用于通过游离半胱氨酸用马来酰亚胺或碘乙酰胺共轭的荧光探针标记蛋白质或肽。

材料和试剂

  1. 巯基反应性马来酰亚胺探针(Life Technologies,Invitrogen TM)
    注意:可从Invitrogen获得大量的探针。
  2. 三 - (2-羧乙基)膦(TCEP)(Life Technologies,Invitrogen TM,目录号:T2556)
  3. 二硫苏糖醇(DTT)(Life Technologies,Molecular Probes,目录号:D1532)
  4. DMSO
  5. 溶解缓冲液(参见配方)

设备

  1. 铝箔
  2. Sephadex G-25列

程序

  1. 在室温下溶解50-100μM的蛋白质于含有1mM TCEP的溶解缓冲液中以保持反应性半胱氨酸减少。
    注意:在与碘乙酰胺或马来酰亚胺缀合期间,不必除去过量的TCEP。 TCEP可以用DTT代替。如果使用DTT,则在引入活性染料前需要透析以除去过量的DTT。
  2. 在使用前立即在DMSO中制备1-10mM硫醇反应性探针的储备溶液。通过将容器包装在铝箔中来保护储存溶液不受光照。
  3. 将巯基反应性探针和蛋白质以约10:1的摩尔比混合,并使反应优选在黑暗中在室温下进行2小时或在4℃下过夜。
    注意:将探针逐滴加入蛋白质溶液中,并搅拌。
  4. 反应完成后,在凝胶过滤柱(例如Sephadex G-25柱)上或通过在4℃下在合适的缓冲液中充分透析,从过量染料中分离偶联物。 >

食谱

  1. 溶解缓冲液(pH 7.0-7.5)
    10-100mM磷酸盐 Tris
    HEPES

参考文献

  1. Hermanson,G.,(1996)。 Bioconjugate Techniques,Academic Press。
  • English
  • 中文翻译
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yan, Q. (2011). Labeling Protein with Thiol-reactive Probes. Bio-protocol Bio101: e82. DOI: 10.21769/BioProtoc.82;
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Yuanqing Lin
One question: In some protocols (very similar to yours), it's recommended as a final step (prior to separation on clumn) to add an excess of Gluthation or BetaMercaptoEthanol, to ensure no reactive species are present.
You do not recommended this step? Why?
6/27/2011 6:49:57 PM Reply
bio-protocol

Adding chemicals with thiol group, such as reduced glutathione and 2-Mercaptoethanol before the column separation of excess dye is optional. Gel filtration method can completely remove excess thiol-reactive reagent. The presence of reactive species have no effect on the label efficiency as well labeled protein.

7/10/2011 1:31:35 AM