Alternatives to MYC epitope tag

Steve Miller Sgmiller at netcom.com
Thu Nov 17 12:17:51 EST 1994


In Article <9411171901.AA2891 at pho018.sb.com>, Seth_M_Fisher%notes at sb.com
(Seth M Fisher) wrote:

>My experience with the FLAG epitope for purification from E. Coli has not been 
>good.
> The binding capacity of the M2 resin was less than 100 ug/ml. This would have
>been OK if the antibody did not also recognize several other proteins, most 
>notably
>chaperonin.
>Also, I could not cleave the FLAG off with enterokinase. The antibody did work
>great for Westerns.
>We switched to a hexa-his tag with more favorable results.
>I would be interested in hearing in a little more detail of some success 
>stories with the
>FLAG technology.
>
    In contrast to your experience, I have had good success using the FLAG
epitope with several proteins. It may be dependent on the exact context. I
have not used FLAG in bacteria, but have used it effectively in both
mammalian cells and insect cells. I have found the M2 gel is capable of
binding at least 1 mg/ml for some constructs and have purified secreted
proteins to homogeneity in a single step from medium containing 10% serum.
Elution can be carried out at neutral pH in low salt by competing off with
the FLAG peptide. The peptide can then be easily removed by diafiltration.
Enterokinase cleavage may again be dependent on the context in which the
FLAG is placed. I have had great luck with Westerns, sensitive and low
background. In addition, the antibodies work well for immunofluorescence and
FACS. Is the chaperonin binding directly to your M2 gel or interacting with
your recombinant protein?



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