Reim D F, Speicher D W
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Anal Biochem. 1992 Nov 15;207(1):19-23. doi: 10.1016/0003-2697(92)90493-q.
The influence of different types of polyvinylidene difluoride (PVDF) membranes on gas phase sequence performance has been evaluated. These PVDF membranes have been classified as either high retention (Trans-Blot and ProBlott) or low retention membranes (Immobilon-P) based on their ability to bind proteins during electroblotting from gels. Initial yields, repetitive yields, and extraction efficiency of the anilinothiazolinone amino acid derivatives have been compared for several standard proteins that have been either electroblotted or loaded onto PVDF membranes by direct adsorption. These results show that the major differences in initial sequence yields between membranes arise from differences in the amount of protein actually transferred to the membrane rather than sequencer-related factors. In contrast to several previous observations from other laboratories, more tightly bound proteins do not sequence with lower initial yields and initial yields are not affected by the ratio of surface area to protein. The stronger binding on high retention PVDF membranes does not adversely affect recoveries of difficult to extract, or very hydrophobic, amino acid derivatives. Several amino acids, especially tryptophan, are actually recovered in dramatically higher yield on high retention membranes compared with either Immobilon or glass filters. At the same time, the protein and peptide binding properties of high retention membranes will frequently improve the repetitive yield by minimizing sample extraction during the sequencer cycle. Stronger protein binding together with improved electroblotting yields offer substantially improved sequence performance when high retention PVDF membranes are used.
已评估了不同类型的聚偏二氟乙烯（PVDF）膜对气相测序性能的影响。这些PVDF膜根据其在从凝胶进行电转印过程中结合蛋白质的能力，被分类为高保留型（Trans-Blot和ProBlott）或低保留型膜（Immobilon-P）。对于通过电转印或直接吸附加载到PVDF膜上的几种标准蛋白质，比较了苯胺噻唑啉酮氨基酸衍生物的初始产率、重复产率和提取效率。这些结果表明，膜之间初始测序产率的主要差异源于实际转移到膜上的蛋白质量的差异，而非与测序仪相关的因素。与其他实验室之前的一些观察结果相反，结合更紧密的蛋白质并非以较低的初始产率进行测序，且初始产率不受表面积与蛋白质比例的影响。高保留型PVDF膜上更强的结合不会对难以提取或非常疏水的氨基酸衍生物的回收率产生不利影响。与Immobilon或玻璃滤器相比，在高保留型膜上，几种氨基酸，尤其是色氨酸，实际上的回收率显著更高。同时，高保留型膜的蛋白质和肽结合特性通常会通过在测序仪循环期间最小化样品提取来提高重复产率。当使用高保留型PVDF膜时，更强的蛋白质结合以及提高的电转印产率可显著改善测序性能。
