Construction of multiple-epitope tag sequence by PCR for sensitive Western blot analysis.
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.
Nakajima
K
K
Department of Molecular Neurobiology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183, Japan.
Yaoita
Y
Y
D015153:Blotting, Western / Q000379:methods*; D017931:DNA Primers / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid; D015843:Serpins / Q000737:chemistry / Q000235:genetics; D014764:Viral Proteins
D017931:DNA Primers; D018076:DNA, Complementary; D000939:Epitopes; D016271:Proto-Oncogene Proteins c-myc; D011994:Recombinant Proteins; D015843:Serpins; D014764:Viral Proteins; C074641:interleukin-1beta-converting enzyme inhibitor