Marlow G C, Wurst D E, Loschke D C
Department of Plant Pathology, Institute of Food and Agricultural Sciences, University of Florida, Gainesville 32611.
Electrophoresis. 1988 Nov;9(11):693-704. doi: 10.1002/elps.1150091104.
Although use of ultrathin polyacrylamide gel isoelectric focusing in the first dimension of two-dimensional analysis bestows a number of advantages, it has been little used by the plant science community. Nonstandardization along with problems unique to the format have probably delayed wider adoption. Relevant parameters were therefore tested in order to optimize resolution, reproducibility, economy and ease of use. Ultrathin-layer gels (200 microns in this study) used in the first dimension require a semirigid backing for support. Widely available matte-finished thin polyester film without chemical pretreatment was found to bind the gel adequately. The gel adheres to the film through all processing steps, yet, if desired, can be easily transferred to Whatman 3MM paper for special applications such as Western blotting. The ultrathin first-dimensional gels can be quickly dried on the polyester backing for convenient handling and long term storage. Strips cut from the dried gel for use in the second dimension are more easily manipulated than their tube format counterparts. The difficulty of disrupting and recovering microsamples of labeled leaf and root tissue prompted the invention of an efficient and simple communition device. An economical and efficient silver stain process is also described. This analytical technique was applied in an attempt to detect resistance gene products in different genetic backgrounds of maize. Although the ultrathin flatbed format provides as good as or better resolution than the tube gel system, the level of sensitivity was still inadequate to reveal the apparently rare resistance gene product.
尽管在二维分析的第一维中使用超薄聚丙烯酰胺凝胶等电聚焦具有许多优点，但植物科学界对其使用较少。格式的非标准化以及该格式特有的问题可能延迟了其更广泛的采用。因此，对相关参数进行了测试，以优化分辨率、重现性、经济性和易用性。第一维中使用的超薄层凝胶（本研究中为200微米）需要半刚性支撑物来支撑。发现广泛可得的未经化学预处理的哑光薄聚酯薄膜能充分粘结凝胶。凝胶在所有处理步骤中都能粘附在薄膜上，但如果需要，也可以很容易地转移到沃特曼3MM纸上用于特殊应用，如蛋白质印迹法。超薄的第一维凝胶可以在聚酯支撑物上快速干燥，便于处理和长期保存。从干燥凝胶上切下用于第二维的条带比管状凝胶对应物更易于操作。破坏和回收标记的叶片和根组织微样品的困难促使发明了一种高效且简单的粉碎装置。还描述了一种经济高效的银染方法。应用这种分析技术试图检测玉米不同遗传背景中的抗性基因产物。尽管超薄平板格式提供了与管状凝胶系统相当或更好的分辨率，但灵敏度水平仍然不足以揭示明显罕见的抗性基因产物。
