Robishaw J D, Balcueva E A
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
Anal Biochem. 1993 Feb 1;208(2):283-7. doi: 10.1006/abio.1993.1047.
The production of antibodies against synthetic peptides derived from amino acid sequences common or unique to a particular protein(s) is an important tool in the identification of structurally related members of an ever-increasing number of protein families. The successful use of anti-peptide antibodies requires that the protein(s) of interest be properly transferred and fully denatured prior to detection by Western-type immunoblotting. In this paper, we demonstrate that conventional transfer procedures are not successful in presenting the G protein gamma subunits in a suitable state for immunodetection. We describe a high temperature (70 degrees C) transfer procedure that results in a more than 20-fold enhancement in the sensitivity of immunodetection of the various G protein gamma subunits. The effect of high temperature transfer could not be duplicated by including 0.2% SDS in the buffer during transfer to nitrocellulose, or by baking or autoclaving the nitrocellulose after transfer. Thus, high temperature transfer is a powerful procedure for enhancing immunoblot detection of protein(s) that may be resistant to denaturation and/or subject to renaturation during the transfer and/or binding to nitrocellulose.
针对源自特定一种或多种蛋白质共有的或独特的氨基酸序列的合成肽产生抗体，是鉴定越来越多蛋白质家族中结构相关成员的一项重要工具。成功使用抗肽抗体要求在通过蛋白质免疫印迹法检测之前，将感兴趣的蛋白质进行适当转移并完全变性。在本文中，我们证明传统转移程序无法成功使G蛋白γ亚基处于适合免疫检测的状态。我们描述了一种高温（70摄氏度）转移程序，该程序可使各种G蛋白γ亚基的免疫检测灵敏度提高20多倍。在转移至硝酸纤维素膜的缓冲液中加入0.2% SDS，或在转移后烘烤或高压灭菌硝酸纤维素膜，均无法重现高温转移的效果。因此，高温转移是一种强大的程序，可增强对在转移和/或与硝酸纤维素膜结合过程中可能抗变性和/或发生复性的蛋白质的免疫印迹检测。
