FLAG tag properties and uses

FLAG tags are octapeptide tags that can be encoded within DNA sequences and be positioned at either end of the protein of interest. FLAG tags can be used for affinity purification, co-precipitation and also for detection by western blotting. The FLAG tag is synthetic, and was designed to be small and hydrophilic, to exert minimal effects on the structure of proteins it is linked to.

Anti-FLAG antibodies are efficient for detection in a range of formats. The FLAG tag also contains an intrinsic enterokinase (EKT) cleavage site, for tag removal after it has fulfilled its purpose.

The FLAG tag sequence, from N to C: DYKDDDDK . It has a molecular weight of 1012 Da.

 

Immunoprecipitation of proteins using FLAG tags: the principle

 

A typical FLAG immunoprecipitation is as follows:


  1. Harvest and lyse host cells with expressed FLAG-tagged protein, and clarify by centrifugation

  2. Incubate the lysate with a slurry of FLAG-capture gel beads, centrifuge and wash carefully.

  3. Elute the bound FLAG-tagged protein and its binding partner(s) with a buffer containing the free FLAG-elution peptide DYKDDDDK.

 

Our product range

Oxford Genetics provides a broad range of plasmids containing N-terminal and C-terminal FLAG tags. In some plasmids the tags flank reporter genes, while in others they are positioned adjacent the MCS enabling you to insert your own gene in frame as required. The plasmid structure shown contains a FLAG tag upstream of the MCS, allowing you to position it simply at the N terminus of your gene of interest, and an enterokinase (EKT) site is also provided that allows simple removal of the FLAG tag from the purified protein if you so wish. In this case a BM40 (osteonectin) secretory tag is also included, to allow efficient secretion of your protein from the expressor cells.  Our online catalogue contains a broad range of plasmid structures, accessible via the buttons below.

Our Plasmid Builder facility also provides a simple means for you to design an efficient strategy for any modifications or cloning manipulations to these plasmids. Finally, as always, although we have designed these plasmids for simple and efficient cloning, we are happy to undertake any cloning steps that you prefer to outsource. You can access all these options through the Plasmid Builder button below.