Applicability of multigene family-specific antibodies toward studies of the subtilases in Arabidopsis thaliana

Polyclonal antibodies were made to two synthetic peptides with sequences patterned after conserved regions in a multigene family of 56 subtilisin-related proteolytic enzymes in Arabidopsis thaliana. GST-fusion proteins encompassing full-length or partial cDNAs bearing putative epitope regions were cloned and expressed in Escherichia coli. Immunoblots showed that the antibodies bound 12 of 13 fusion proteins tested. About 27 more subtilase genes code for regions with sequences very similar to the epitope regions of the subtilases that were visualized on the immunoblots. Subtilases in rosette and cauline leaves, stems, flowers, and siliques could be distinguished by the antibodies; some binding the two antibodies to similar extents, while others bind preferentially or almost exclusively to one or the other antibody. When antibodies were used to monitor ion-exchange fractionation of seedling extracts, one specific subtilase was identified by LC–MS–MS. The specificity of the antibodies extended to subtilases in soybean. These studies show that multigene family-specific antibodies can be applied to the study of gene families, where sequence similarities make it difficult to produce antibodies specific for each individual protein in the group.