Reaction electrophoresis

The differential mobilities of compounds in an electric field are important analytical criteria and we can use them to bring electrophoretically pure components of a mixture medium on which they are separated. To this end, the compound undergoing reaction are brought into positions on the carrier to assure optimal contact between selected fractions, within a predetermined domain of time and distance. The appearance of a product defines their reactivities, and the product's continued migration on the same carrier can provide the first key to its identity as is demonstrated and discussed. The method is called reaction electrophoresis and it will be of particular use in studies with labile components. It is illustrated here with the coupling reaction of the sodium salt of 1,4-naphthol sulfonic acid and tetrazotized o-dianisidine.     

Plastein Reaction

α-Glucosidase (α-d-glucoside glucohydrolasc; EC: 3.2.1.20) has been extracted from bovine spleen and separated into two fractions (Fractions A and B) by gel filtration on Sephadex G-200. Fraction B which was retarded on Sephadcx columns contained only an acid α-glucosidase. This enzyme catalyzed hydrolysis of maltose and glycogen at comparable rates. Glycogen was hydrolyzed almost completely to glucose. The results of heat-treatment and of inhibition bv turanose demonstrated that Fraction A contained both acid if and neutral α-glucosidases. The neutral enzyme in Fraction A was further separated into four different components on preparative polyacrylamide gel electrophoresis. All of these neutral enzymes showed similar catalytic properties each other, and hydrolyzed maltose much more rapidly than glycogen. The acid enzyme in Fraction A was inactivated on the electrophoresis and was not further characterized.     

PCR法

PCR法自1985年公布后一下子就成为了一项基础技术。能够从极微量的DNA中进行扩增的PCR法，也开始向RNA研究以及定量分析方面发展。现在也被广泛用于诊断、动植物育种等实用过程中。此讲由宝生物制品开发中心主任向井博之执行董事讲解。[编者按]     

基于SOS反应及氧化应激反应相关启动子的辐射生物传感器研究

近年来的动物实验结果表明电磁辐射的危害主要是具有神经系统毒性、诱发肿瘤和生殖系统损伤等,广域、隐蔽和累积效应是辐射的特点,除对机体进行直接损伤外,还可导致间接损伤,即通过产生活性氧(ROS)和自由基攻击生物大分子。为了迅速和简便地检测辐射毒性的大小建立了新型的辐射生物传感器,构建了携带SOS反应和氧化应激反应相关的SulA、RecA、Cda和SoxR四种启动子融合经过密码子简并性优化的增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告因子的工程菌传感器,并对这些生物传感器进行了γ射线辐照处理,筛选出了针对γ射线响应较好的,优选RecA工程菌传感器。利用PCR和Overlap PCR克隆获得了启动子-报告因子融合基因,并插入表达载体PUC19中,转化入宿主大肠杆菌DH5α,通过提取质粒进行双酶切和测序验证后,将构建成功的工程菌传感器首先进行化学毒性试剂刺激,一旦化学试剂刺激结果阳性便进行物理辐射刺激。结果显示,构建成功的4种工程菌传感器均对物理辐射产生应答,且随物理辐射剂量的增加(0～30Gy),绿色荧光强度逐渐增强。运用合成生物学手段,成功建立基于生物损伤修复效应和氧化应激反应的辐射生物传感器,具有制备简便、结果可视性等优点,能满足快速、广范围、在线监测的需求,在细胞毒性物、辐射环境乃至空间射线的损伤能力测定方面具有良好的应用前景。