Chameleon labels for staining and quantifying proteins
Chameleon labels for staining and quantifying proteins
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DOI：
10.1002/anie.200460508
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发表时间：
2004-01-01
影响因子：
16.6
通讯作者：
Wolfbeis, OS
中科院分区：
文献类型：
--
作者：
Wetzl, BK;Yarmoluk, SM;Wolfbeis, OS
分析蛋白质模式、其时间变化以及解释其功能是目前令人着迷的技术之一，通常被称为蛋白质组学。[1]蛋白质模式可以通过多种方法进行分析，包括凝胶电泳、印迹或所谓的生物芯片。[2]虽然生物芯片（和蛋白质阵列）优选地应用于已知蛋白质组成的系统以鉴定特定蛋白质，但是一维或二维形式的电泳容易地应用于未知样品。[2]由于凝胶电泳是一种分离技术，它还需要适当的方法来测定（或可视化）蛋白质。[3]用于可视化的标准方法包括银染色或用染料染色，例如考马斯亮蓝（CB B）或酰胺黑B。[4]用于蛋白质的染色和可视化的荧光方法是特别感兴趣的，这是因为荧光方法的高灵敏度。（激光诱导）荧光，已达到纳米和皮摩尔（如果不是zeptomole或单分子）水平，至少对于溶液而言。需要区分两种类型的荧光蛋白染色剂：第一个涉及染色剂与蛋白质的官能团（如氨基或巯基）的共价连接，第二个涉及非共价蛋白质-染色剂相互作用。两者各有千秋。与蛋白质的共价连接是稳定的（即标签不能被洗掉），而非共价标记使得例如质谱能够进行，因为染色时蛋白质的总质量没有变化。[5]典型的非共价蛋白质染色剂包括SYPRO染料（某些有机或有机金属荧光染料），它们发出红色或粉红色的光。[6]染色剂以高亲和力与蛋白质结合，然后可以在凝胶中以2-10 ng/条带的量测定。相反，共价荧光标记广泛用于聚丙烯酰胺凝胶电泳（PAGE）。通过预染色（即电泳前）或电泳后实现缀合。已知多种共价结合标记。[6-8]然而，所有这些都具有在游离和蛋白质中相同的光谱特性（在几nm内）。
The analysis of a protein pattern, its temporal changes, and the interpretation of its function is one of the fascinating technologies at present and is often referred to as proteomics.[1] Protein patterns can be analyzed by a variety of methods including gel electrophoresis, blotting, or by socalled biochips.[2] While biochips (and protein arrays) are preferably applied to systems of known protein composition to identify specific proteins, electrophoresis in one-or twodimensional form is readily applied to unknown samples.[2] Since gel electrophoresis is a separation technique, it also requires appropriate methods for determination (or visualization) of proteins.[3] Standard methods for visualization include silver staining or staining with dyes, such as Coomassie Brilliant Blue (CBB) or Amido Black B.[4] Fluorescent methods for staining and visualization of proteins are of particular interest because of the high sensitivity of (laserinduced) fluorescence, which has reached the nano-and picomole (if not zeptomole or single molecule) level, at least for solutions.Two types of fluorescent protein stains need to be distinguished: The first involves covalent linkage of the stain to a functional group of a protein (such as amino or thiol), the second involves noncovalent protein–stain interaction. Both have their merits. A covalent linkage to the protein is stable (ie, the tag cannot be washed out), while noncovalent labeling enables, for example, mass spectroscopy to be performed because no change in the total mass of the protein occurs on staining.[5] Typical noncovalent protein stains include the SYPRO dyes (certain organic or organometallic fluorochromes) that give red or pink emissions.[6] The stains bind to proteins with high affinity which then can be determined in gels in quantities of 2–10 ng/band. Covalent fluorescent labeling, in contrast, is widely used in polyacrylamide gel electrophoresis (PAGE). Conjugation is achieved by either pre-staining (that is, before electrophoresis) or after electrophoresis. A variety of covalently binding labels are known.[6–8] However, all of them have spectral properties that are identical (within a few nm) in the free and the protein-