Epitope tags in Drosophila - summary

Gerard Manning ger at stanford.edu
Wed Oct 2 15:04:06 EST 1996


Here is a summary of the responses I got to my query on the use of eptope
tags in Drosophila, along with some other information I pulled together.
If anyone has more information to contribute, especially on the use of
epitope tags in embryos (whole mounts or Westerns), please post or email.
The updated version of this file will then be placed on the Web,
accessible through the Drosophila Virtual Library at:
   http://www-leland.stanford.edu/~ger/drosophila.html

      -Gerard.




                        Epitope tags in Drosophila

 >FLAG (DYKDDDDK)
Katy Afshar [1] used the M5 antibody (IBI 3091) from Kodak (on Westerns
and confocal stains of ovaries). Other FLAG antibodies may give higher
background, according to a previous posting on this group.

 >myc
This is probably the most widely used epitope tag in general, but I've
heard many rumours that the antibodies against myc are poor or variable.
David Micklem has tagged constructs driven by maternal alpha4 tubulin and
bicoid promoters and got good detection by Western, and fair staining of
ovaries with the 9E10 monoclonal. He reccomends the BAbCo antibody over
that of Oncogene Science, for staining quality and price. Doreen Gillespie
had high background with that antibody (ovaries?). Tian Xu [2] used myc as
a clonal marker which stained well in discs, ovaries and adult frozen
sections. Kramer & Phistry [3] got clean staining with 9E10 on Westerns of
S2 cells. Carol Schwartz and others in Tom Kornberg's lab [4] have used a
mAb from Mike Bishop's lab to successfully stain discs and cell lines.

 >Hemagglutinin (HA) [(M)YPYDVPDYA(SS)]
Mike Forte detected HA cleanly with the 12CA5 antibody from Boehringer or
BAbCO in imaginal discs and pupal wings. Kramer & Phistry [3] got clean
staining with 12C15 on Westerns of S2 cells. Surdej and Jacobs-Lorena [5]
have created up a useful cassette for inserting a HA tag at many sites in
a protein, and found clean staining, by Western, in ovaries and early
embryos. They also report that 12CA5 has been used for Westerns,
immunofluroesence and immunoprecipitation.


 >References
[1] Ashfar et al (1995) Identification of the chromosome localization
domain of the Drosophila Nod kinesin-like protein. J. Cell Biol. 130
833-843.

[2] Xu & Rubin (1993) Analysis of genetic mosaics in developing and adult
Drosophila tissues. Development 117: 1223-37

[3] Kramer & Phistry (1996) Mutations in the Drosophila hook gene inhibit
endocytosis of the boss transmembrane ligand into multivesicular bodies. J
Cell Biol. 133: 1205-15

[4] Tabata et al (1995) Creating a Drosophila wing de novo, the role of
engrailed, and the compartment border hypothesis. Development 121: 3359

[5] Surdej & Jacobs-Loena (1994) Strategy for Epitope Tagging the
Protein-Coding Region of Any Gene. Biotechniques 17: 560-565



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