The detection and analysis of protein isoforms is a complicated task especially if they differ only in small specific domains. Obtaining specific polyclonal antibodies against these domains is a challenge, but if successful it can have a wide range of applications, such as in proteomics and immunochemical analysis. We show herein a method of overexpression and purification of two small specific domains corresponding to the isoforms b and c of the murine transcription factor Pitx2, and the generation and purification of monospecific polyclonal antibodies against them, by using a two-step affinity purification procedure, based on the use of CNBr-Sepharose matrix. Such a method also allows recovering monospecific polyclonal antibodies against the tag fusion peptide (C-LYTAG tag). The specificity of the isolated polyclonal antibodies was demonstrated by Western blot and immunohistochemical analysis. In addition, our protocol is easily scalable and allows the generation of monospecific polyclonal antibodies for large-scale analysis. (c) 2008 Elsevier Inc. All rights reserved.