Related Experiment Video
Updated: Jul 5, 2026
Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag
Published on: January 16, 2012
Expression in bacteria of small and specific protein domains of two transcription factor isoforms, purification and
Francisco Hernández-Torres1, José Rafael Pedrajas, Amelia E Aránega
1Departamento de Biología Experimental, Facultad de Ciencias Experimentales, Universidad de Jaén, Paraje de las Lagunillas, s/n, 23071 Jaén, Spain.
Abstract:
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.
