DeCaprio James, Kohl Thomas O
Cold Spring Harb Protoc. 2019 Feb 1;2019(2):2019/2/pdb.prot098624. doi: 10.1101/pdb.prot098624.
This protocol describes the cross-linking of antibodies to either Protein A or G agarose beads using dimethyl pimelimidate (DMP). DMP contains an imidoester at each end of a 7-carbon spacer arm and forms an amidine bond with amino groups at alkaline pH; however, cross-linking is more efficient when performed at pH >8. DMP will react with primary amines; thus, it is important that the cross-linking procedure is conducted using nonamine-containing buffers. Following the antibody-bead incubation, beads are washed in Borate buffer to remove residual amines from the Tris buffer. After completion of the cross-linking process in the presence of DMP, unreacted DMP is quenched with ethanolamine, and beads are washed extensively to remove residual noncross-linked antibody before immediate use or storage at 4°C.
本方案描述了使用二甲基庚二酸亚胺酯（DMP）将抗体与蛋白A或G琼脂糖珠交联的方法。DMP在7碳间隔臂的两端各含有一个亚胺酯，在碱性pH条件下与氨基形成脒键；然而，在pH>8时进行交联效率更高。DMP会与伯胺反应；因此，使用不含胺的缓冲液进行交联过程很重要。抗体与珠子孵育后，珠子在硼酸盐缓冲液中洗涤，以去除Tris缓冲液中的残留胺。在DMP存在下完成交联过程后，未反应的DMP用乙醇胺淬灭，珠子在立即使用或在4°C储存前进行广泛洗涤以去除残留的未交联抗体。
