tag for purification... - wich one do you prefer ? (Jul/21/2007 )
facing the problem in my lab, i wanted to start a topic.
I have nice purifs with flag, but bad with ha...
so which one do you prefer ?
you may add somme comments if you want. would be nice.
if you have discovered useful tips, please put them in comments.thank you
NB : please pm if you want that i add a particular flag, or don't hesitate to edit the topic.
be careful when using the HA-tag as it has recently been shown to be cut during apoptosis.
http://www.ncbi.nlm.nih.gov/sites/entrez?h...p;term=17264856
I prefer to use the myc-tag, but that's mainly because we purify this antibody in-house making it the cheapest one. The flag-tag, I don't like it for IF, but for IP I have used it quite succesfully (although the alleged co-immunoprecipitating protein wouldn't cooperate)
For purification, i am not too familiar which is better.
For western blots, I would go with HA tag and for immunofluorescence I would go with AU1. Not many might have heard about this tag. But we have tried to find the one suitable for invivo and found this was the best out of 6 different tags.
I have attached a paper which you can check for the different tags we tested but we do not have any data for purifications.
[attachment=3359:Tags_paper.pdf]
http://www.ncbi.nlm.nih.gov/sites/entrez?h...p;term=17264856
I prefer to use the myc-tag, but that's mainly because we purify this antibody in-house making it the cheapest one. The flag-tag, I don't like it for IF, but for IP I have used it quite succesfully (although the alleged co-immunoprecipitating protein wouldn't cooperate)
Could you tell us what type of anti-myc antibody is that, monclonal or polyclonal, where did it originate from?
the anti-myc antibody which is purified in our institution is the mouse monoclonal anti-myc antibody from the 9E10 clone. I don't know where we obtained the cells, but you can order them for instance using the Developmental Studies Hybridoma Bank (http://dshb.biology.uiowa.edu/commerce/ccp1061-c-myc-9e-10.htm).
I have nice purifs with flag, but bad with ha...
so which one do you prefer ?
you may add somme comments if you want. would be nice.
if you have discovered useful tips, please put them in comments.thank you
NB : please pm if you want that i add a particular flag, or don't hesitate to edit the topic.
why is GFP and its derivatives are missing?
I prefer Flag. I always had better results using it.
Anyway, I think that His-tag and Tap-tag (if you look for associates) are always the best choices in my opinion.
Good luck
i meant by this poll using an epitope tag for pruduction and purification of protéins, and associated, cellular/tissue sublocalization and WB.
that's wh i didn't wrote the GFP.
i meant by this poll using an epitope tag for pruduction and purification of protéins, and associated, cellular/tissue sublocalization and WB.
that's wh i didn't wrote the GFP.
you may enrich GFP-tagged proteins, including IP, with anti-GFP; we routinely use a GFP-IP- kit form Miltenyi Biotech;
it is not as elegant as the other polled methods, but GFP is more flexible to use (IP, IB, ICC/IHC)
ok
i implemented with GFP and 6his tags