Producing A Flag-Tagged Protein

Biochemistry, Molecular Biology, and Cell Biology Protocols >> Producing a Flag-Tagged Protein

1. Make sure that the sequence of your gene of interest and the gene sequence of the Flag tag do not contain any of the restriction sites that you choose to ligate into your vector with.
2. Perform PCR of the gene that you wish to fuse with the Flag tag. If attaching the Flag tag to the C-terminus of the protein, remember to remove the stop codon from the gene when designing your primer, and put the stop codon after the flag tag. If attaching the Flag tag to the N-terminus, it is probably better to attach it several codons downstream from the start codon rather than right after the start codon. Your forward primer will usually be a sequence from your gene or the DNA sequence of the Flag tag, and the reverse primer will usually be the sequence from the Flag tag or your gene, respectively. Remember to generate restriction sites on either site of the PCR product so that you can ligate it into a suitable vector.
3. Digest out the restriction sites from a vector, and purify out the vector to remove the DNA between the restriction sites. After completing the PCR of the Flag-tag fused gene, also digest the PCR product with the restriction sites that had been designed at both ends of the gene.
4. Perform a ligation with the PCR product containing your gene attached to the DNA sequence of the flag tag, and the target vector.
5. Transform the desired amount of ligated product into competent bacterial cells.
6. Select the desired colonies and grow up the bacteria in overnight cultures.
7. Purify the plasmid (eg can use commercial kits).
8. Store the purified plasmid at -80 degrees C or -20 degrees C in aliquots.
9. Express the flag-tagged gene as a protein either in bacteria such as E. coli via induction, or in higher-level organisms such as mammals, where you can perform transfection.
10. If desired, purify the expressed flag-tagged protein.