一种用于亚微克负载水平弗格森图分析的薄层多带琼脂糖凝胶电泳装置。

A thin-layer multistrip agarose gel electrophoresis apparatus for Ferguson plot analysis at the sub-microgram load level.

作者信息

Orbàn L, Sullivan J V, Chrambach A

机构信息

Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, Bethesda, MD 20892.

出版信息

J Biochem Biophys Methods. 1989 Jul;19(1):105-20. doi: 10.1016/0165-022x(89)90055-9.

DOI:10.1016/0165-022x(89)90055-9

PMID:2809063

Abstract

A method for the simultaneous horizontal agarose gel electrophoresis on thin-layer strips of different gel concentrations was developed for the purpose of generating Ferguson plots at the sub-microgram load level. Seven independent gel strips on a common GelBond support were formed by filling channels created by a comb-shaped spacer (polycarbonate) in a vertical multistrip cassette. Electrophoresis on a horizontal Peltier-cooled surface employed commercial apparatus (E-C Apparatus Corp.) with a modified cover which is airtight and holds anodic and cathodic voltage measurement probes for each strip. The application of the apparatus to Ferguson plot analysis in a single experiment was exemplified on the RNA-containing turnip crinkle virus (TCV) at a load of 50 ng/gel strip, using an optimized silver staining method (a modification of a procedure of FMC Corp. BioProducts) for detection. Within the range of 3.5 to 12.5 V/cm, the plot was found to be independent of field strength. Mobility is also independent of the concentration of detergent (CHAPS) up to 10 mM.

摘要

为了在亚微克负载水平生成弗格森图，开发了一种在不同凝胶浓度的薄层条带上同时进行水平琼脂糖凝胶电泳的方法。通过在垂直多带盒中填充由梳状间隔物（聚碳酸酯）形成的通道，在普通的GelBond支持物上形成七条独立的凝胶条带。在水平珀耳帖冷却表面上进行电泳时，使用商业仪器（E-C仪器公司），该仪器带有一个经过改进的盖子，该盖子是气密的，并为每个条带持有阳极和阴极电压测量探针。以负载量为50 ng/凝胶条带的含RNA的芜菁皱缩病毒（TCV）为例，在单个实验中将该仪器应用于弗格森图分析，并使用优化的银染方法（FMC公司生物产品程序的改进方法）进行检测。发现在3.5至12.5 V/cm的范围内，该图与场强无关。在高达10 mM的范围内，迁移率也与去污剂（CHAPS）的浓度无关。