Reim D F, Speicher D W
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Anal Biochem. 1993 Oct;214(1):87-95. doi: 10.1006/abio.1993.1460.
An optimized sequencer program with a cycle time of 38 min which is specifically tailored for analysis using polyvinylidene difluoride (PVDF) membranes has been developed. The program was developed using a pulsed liquid-phase instrument which was converted to gas-phase acid delivery. Gas-phase acid delivery minimized sample extraction from PVDF membranes and improved tryptophan yields in at least some cases. Other modifications which contributed to reliable high sensitivity sequencer performance included use of a Blott cartridge, substitution of ethyl acetate:heptane (1:1, v/v) instead of butyl chloride as the extraction solvent, use of a modified 100-microliters injection loop with an internal restrictor to reliably inject nearly 90% of the sample, and an HPLC gradient which resolved tryptophan from diphenylurea. These shortened cycle times were achieved at the conventional gas-phase reaction temperature. A slight increase in lag or carryover at prolines was compensated by reduced background from nonspecific acid cleavage which facilitated extended and/or high sensitivity sequencing of large proteins. Reproducible high initial and repetitive cycle yields were obtained with a wide range of experimental peptides which were electroblotted from either 1D or 2D polyacrylamide gels onto high retention PVDF membranes. Initial yields of the majority of the experimental samples analyzed with this program were less than 5 pmol. In addition, most samples with initial yields below 1-2 pmol yielded sufficient sequence information to identify the protein by comparison to protein sequence data-bases or to design oligonucleotide probes.
已开发出一种优化的测序程序，其循环时间为38分钟，该程序是专门为使用聚偏二氟乙烯（PVDF）膜进行分析而定制的。该程序是使用脉冲液相仪器开发的，该仪器已转换为气相酸输送。气相酸输送在至少某些情况下可最大程度减少从PVDF膜中提取样品，并提高色氨酸产量。有助于实现可靠的高灵敏度测序仪性能的其他改进包括使用Blott柱、用乙酸乙酯：庚烷（1:1，v/v）代替丁基氯作为萃取溶剂、使用带有内部限流器的改良100微升进样环以可靠地注入近90%的样品，以及一种能将色氨酸与二苯基脲分离的HPLC梯度。这些缩短的循环时间是在传统的气相反应温度下实现的。脯氨酸处滞后或残留的轻微增加通过非特异性酸裂解产生的背景降低得到补偿，这有助于对大蛋白质进行延长和/或高灵敏度测序。从1D或2D聚丙烯酰胺凝胶电印迹到高保留PVDF膜上的各种实验肽都获得了可重复的高初始循环产率和重复循环产率。用该程序分析的大多数实验样品的初始产率低于5皮摩尔。此外，大多数初始产率低于1 - 2皮摩尔的样品产生了足够的序列信息，可通过与蛋白质序列数据库比较来鉴定蛋白质或设计寡核苷酸探针。
