Hilpert Kai, Winkler Dirk F H, Hancock Robert E W
Centre for Microbial Diseases and Immunity Research, University of British Columbia, #2259 Lower Mall Research Station, Vancouver, British Columbia, Canada V6T 1Z3.
Nat Protoc. 2007;2(6):1333-49. doi: 10.1038/nprot.2007.160.
Peptide synthesis on cellulose using SPOT technology allows the parallel synthesis of large numbers of addressable peptides in small amounts. In addition, the cost per peptide is less than 1% of peptides synthesized conventionally on resin. The SPOT method follows standard fluorenyl-methoxy-carbonyl chemistry on conventional cellulose sheets, and can utilize more than 600 different building blocks. The procedure involves three phases: preparation of the cellulose membrane, stepwise coupling of the amino acids and cleavage of the side-chain protection groups. If necessary, peptides can be cleaved from the membrane for assays performed using soluble peptides. These features make this method an excellent tool for screening large numbers of peptides for many different purposes. Potential applications range from simple binding assays, to more sophisticated enzyme assays and studies with living microbes or cells. The time required to complete the protocol depends on the number and length of the peptides. For example, 400 9-mer peptides can be synthesized within 6 days.
使用SPOT技术在纤维素上进行肽合成,能够以少量并行合成大量可寻址的肽。此外,每条肽的成本不到传统在树脂上合成肽成本的1%。SPOT方法遵循常规纤维素片上的标准芴甲氧羰基化学原理,并且可以使用600多种不同的构件。该过程包括三个阶段:纤维素膜的制备、氨基酸的逐步偶联以及侧链保护基团的裂解。如有必要,可将肽从膜上裂解下来,用于使用可溶性肽进行的检测。这些特性使该方法成为用于多种不同目的筛选大量肽的出色工具。潜在应用范围从简单的结合检测到更复杂的酶检测以及对活微生物或细胞的研究。完成该方案所需的时间取决于肽的数量和长度。例如,400条9肽可以在6天内合成。