Enhanced gel mobility shift assay for DNA-binding factors.
Enhanced gel mobility shift assay for DNA-binding factors.
复制标题
DOI：
10.1006/abio.1993.1400
复制
发表时间：
1993-08
影响因子：
2.9
通讯作者：
H. Hassanain;W. Dai;S. Gupta
中科院分区：
文献类型：
--
作者：
H. Hassanain;W. Dai;S. Gupta
凝胶迁移率改变分析通常被用来研究DNA结合因子，这些因子参与了构成基因、组织特异性基因和诱导基因的调节。我们发现，在细胞提取物中加入相对较高浓度(2.5%或更高)的3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonate(CHAPS，一种两性离子洗涤剂)对四种不同因子(AP-1、SP1、GATA-1和干扰素α调节因子ISGF3)的DNA结合反应极大地增强了DNA-蛋白质复合体的信号(最高可达20倍)。这样获得的DNA-蛋白质复合体的放大信号是(至少部分)由于结合效率的提高，正如凝胶中自由探针的数量大大减少所揭示的那样。然而，结合的特异性并没有受到影响。CHAPS对四种不同因子的DNA-蛋白质复合体的形成有促进作用，提示CHAPS的促进作用可能更为普遍，并不局限于某些类型的DNA结合因子。这些结果为DNA结合因子的高灵敏分析提供了基础，这可能对几种类型的此类因子的研究有用。在被测试的其他洗涤剂中，Chapso(另一种两性离子洗涤剂)、NP-40和辛基葡萄糖苷(非离子洗涤剂)也被发现促进了AP-1结合的复合体的形成，而胆酸钠和脱氧胆酸盐表现出强烈的抑制作用。
Gel mobility shift assays are commonly used to study DNA-binding factors involved in the regulation of constitutive, tissue-specific, and inducible genes. We found that addition of 3-[(3-cholamidopropyl)dimethylammonio]-propanesulfonate (Chaps, a zwitterionic detergent) at relatively high concentration (2.5% or more) to DNA-binding reactions for four different factors (AP-1, SP1, GATA-1, and interferon alpha-regulated factor ISGF3) assayed in cell extracts greatly enhanced the signal for DNA-protein complexes (up to about 20-fold). The amplified signal for DNA-protein complexes so obtained was (at least in part) due to increased binding efficiency, as revealed by greatly reduced amounts of the free probes in the gels. The binding specificity, however, was not compromised. The fact that DNA-protein complex formation with four different factors was stimulated by Chaps suggests that the enhancing effect of Chaps may be more general and not limited to certain types of DNA-binding factors. The results provide the basis for a highly sensitive assay for DNA-binding factors, which may be useful in several types of studies on such factors. Among other detergents tested, Chapso (another zwitterionic detergent), NP-40, and octylglucopyranoside (nonionic detergents) were found also to enhance the complex formation as tested for AP-1 binding, whereas sodium cholate and deoxycholate showed strong inhibition.