A method for immediate visualization of proteins in acrylamide gels and its use for preparation of antibodies to enzymes.
A method for immediate visualization of proteins in acrylamide gels and its use for preparation of antibodies to enzymes.
复制标题
一种立即可视化丙烯酰胺凝胶中蛋白质的方法及其在制备酶抗体中的用途。
DOI：
10.1016/0003-2697(69)90132-8
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发表时间：
1969
影响因子：
2.9
通讯作者：
S. Udenfriend
中科院分区：
文献类型：
--
作者：
Boyd K. Hartman;S. Udenfriend
目前用于定位丙烯酰胺凝胶中的蛋白质的染色方法需要事先将蛋白质固定在酸中，然后染色并去除多余的染料。这些方法除了耗时外，还会使蛋白质完全变性。为了获得未变性的蛋白质，有必要通过与染色凝胶的比较或使用确定的迁移距离来估计所需蛋白质在未染色凝胶中的位置。本文描述了一种在几分钟内染色蛋白质的简单方法，通常在中性pH下，变性最小。由于该方法快速，扩散的分辨率损失很小。染色后，相对未变性的蛋白带可以在直接观察下从凝胶中小心地切下。苯胺萘磺酸盐（Anilinonaphthalene sulfonate， ANS）在水中不发出荧光，但在有机溶剂或各种蛋白质水溶液中溶解时，在可见光范围内发出明亮的荧光(1)。荧光增强可能是由于荧光团与蛋白质表面的疏水位点结合(2)。事实上，大多数蛋白质都可以通过化学或热变性来增强ANS荧光(3)。下面描述的丙烯酰胺凝胶的染色过程利用了ANS的上述特性，并且不可避免地存在一些由凝胶聚合中使用的氧化剂残留物引起的蛋白质带变性，以及一旦凝胶在电泳后从其管中取出后表面变性。1-苯胺- s -萘磺酸盐镁盐（伊士曼有机化学品）从热水中两次重结晶(4)。细小的晶体通过过滤和干燥除去。制备一种原液（1mg /ml），在4℃下稳定至少6个月。的
Staining methods presently available for localizing proteins in acrylamide gels require prior fixation of the protein in acid, followed by staining and removal of excess dye. In addition to being lengthy, these methods cause complete denaturation of the protein. In order to obtain undenatured protein it is necessary to estimate the position of the desired protein in an unstained gel either by comparison to a stained gel or by using established migration distances. This paper describes a simple method for staining proteins within minutes, often at neutral pH, with minimal denaturation. Since the method is rapid, there is little loss of resolution by diffusion. Following staining the relatively undenatured protein band can be carefully cut from the gel under direct observation. Anilinonaphthalene sulfonate (ANS) does not fluoresce in water, but fluoresces brightly in the visible range when dissolved in organic solvents or in aqueous solutions of various proteins (1). The enhanced fluorescence is presumably due to binding of the fluorophore to hydrophobic sites on the protein surface (2). In fact most proteins can be made to enhance ANS fluorescence following chemical or thermal denaturation (3). The staining procedure described below for acrylamide gels utilizes the above properties of ANS and the fact that there is inevitably some denaturation of protein bands caused by remnants of the oxidizing agent used in polymerization of the gel, and by surface denaturation once the gels are removed from their tubes following electrophoresis. The magnesium salt of 1-anilino-S-naphthalene sulfonate (Eastman Organic Chemicals) is twice recrystallized from hot water (4). The fine crystals are removed by filtration and dried. An aqueous stock solution (1 mg/ml) is prepared and is stable at 4 C for at least six months. The