I am making a tag for ATG8 for my western blot analysis. I was able to construct ATG7 and ATG1 tagged with TAP but not with ATG8. I was checking on some papers and noticed that all or almost all of ATG8 was tagged with GFP but not TAP. I know that GFP can be used for fluorescence studies but some GFP-ATG8 papers I searched did not have fluorescence studies, only western blot.
GFP works extremely well not only as a fluorescent marker but also as an immunological tag. Antibodies against GFP are highly specific/robust and very cheap. Furthermore, the GFP tag is cleaved off during maturation of the autophagasome, so the tagged protein is useful for many reasons. These are probably the reasons you don't see other tags (FLAG, myc, etc).
I'm not sure why you are tagging ATG8 yourself - there are good commercially available antibodies to this protein.
I think the reason is that the GFP itself is very stable under proteolytic conditions. So after the autophagosome fused with the lysosome, where most proteins would already be degraded GFP is still visible. Making it an excellent marker for ongoing Autophagy.
Well one good reason is also that GFP is simply more widespread as a tag than TAP, hence you are very much more likely in general to find that your favorite protein has been already reported to being fused to GFP rather than to TAP.
As far as I understood the original version of the TAP-tag was traditionnally fused to the C-terminus of proteins of interest which clearly render its use impossible whith conjugated ATG8, which is usually processed proteolytically at its C-terminus during autophagosome formation. Now N-terminus version has emerged and would potentially be usable with ATG8. The advantage of a TAP-tag is that it allows through its double affinity purification steps to reduce the amount of contaminating proteins present in the eluate containing the protein of interest (ATG8) and its potentially genuine protein interacting partners which are coeluting (see wikipedia link below) .
According to your experience, Simon, which are the best commecially available antibodies againts human ATG8/LC3?
http://en.wikipedia.org/wiki/Tandem_affinity_purification
- Badges
- Science method