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Construction of multiple-epitope tag sequence by PCR for sensitive Western blot analysis
1Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183, Japan.
Abstract:
Epitope tagging is a powerful technique to characterize a recombinantly expressed protein encoded by cDNA without the purification of the protein and the immunization of animals. In some cases, however, the expression of a tagged protein is too low to analyze by Western blot. We have developed a simple method to generate tandem repetitive nucleotide sequence by PCR, which allows us to label a protein of interest with a multiple-epitope tag. When five myc epitopes were attached to vaccinia virus protein CrmA, its signal was multiplied 5.8 times in Western blot analysis, compared with that of one epitope-tagged CrmA.