Liang S P, Laursen R A
Department of Chemistry, Boston University, Massachusetts 02215.
Anal Biochem. 1990 Aug 1;188(2):366-73. doi: 10.1016/0003-2697(90)90622-g.
The use of prepacked capillary columns for immobilizing proteins and peptides for solid-phase Edman degradation is described. Capillary tubes with an internal volume of about 30 microliters are filled with glass beads bearing isothiocyanato groups (DITC-glass), aminophenyl groups (AP-glass), or aminoethylaminopropyl groups (AEAP-glass) and are sealed with porous plugs. Proteins or peptides in appropriate buffers are introduced into the columns by capillary action and are covalently coupled to the glass beads, either by reaction of lysine side-chain amino groups with DITC-glass, by carbodi-imide-mediated reaction of carboxyl groups with AP-glass, or by reaction of homoserine lactone groups with AEAP-glass. Optimization of attachment conditions is described. The capillary columns are loaded into the sequencer and, when sequencing has been completed, are discarded. This technique greatly simplifies polypeptide immobilization and is suitable for microsequencing (less than 50-1000 pmol) or macrosequencing (1-50 nmol).
本文描述了使用预装毛细管柱固定蛋白质和肽用于固相埃德曼降解的方法。内部体积约为30微升的毛细管填充有带有异硫氰酸根基团的玻璃珠（DITC-玻璃）、氨基苯基基团（AP-玻璃）或氨乙基氨丙基基团（AEAP-玻璃），并用多孔塞密封。在适当缓冲液中的蛋白质或肽通过毛细管作用引入柱中，并通过赖氨酸侧链氨基与DITC-玻璃的反应、碳二亚胺介导的羧基与AP-玻璃的反应或高丝氨酸内酯基团与AEAP-玻璃的反应与玻璃珠共价偶联。文中描述了连接条件的优化。将毛细管柱装入测序仪，测序完成后丢弃。该技术极大地简化了多肽固定，适用于微量测序（小于50-1000皮摩尔）或大量测序（1-50纳摩尔）。
