LAUSR

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.