epitope tag: two or one?
chrisb at hgu.mrc.ac.uk
Mon Jun 22 05:58:17 EST 1998
rmeili_nospamare at hotmail.com wrote:
: Thanks for your answers:
: I was hoping for a more than linear effect for two vs one epitope which
: seems not to be the case
According to rumour, sometimes the effect _is_ greater than linear. I
haven't seen this supported in published data, though. And the more
tags you add, the more likely you are to mess up the protein's
structure and function.
: Chris Boyd and Darren Natale: It is cheap to add one epitope but it will be
: more difficult to add multiple epitopes by PCR alone.
Why? You can add 4 HA tags to the C-terminus in one go with a primer
of only (eg) 20 [for initial priming] + 4*27 [the tags] + "TGAATTC"
[stop codon + EcoRI site] + 1 [for luck] = 132 nt: even long oligos
are not expensive these days. You can eliminate the possibility of
mispriming by creative choice of wobble position bases in the tags.
Admittedly, after PCR you MUST sequence to verify the constructs, but I
still think it's more cost-effective than using tagging vectors (except
perhaps for one-offs).
Chris Boyd | from, but not \ MRC Human Genetics Unit,
Christopher.Boyd at hgu.mrc.ac.uk | on behalf of / Western General Hospital,
http://www.hgu.mrc.ac.uk/Users/Christopher.Boyd \ Edinburgh, EH4 2XU, SCOTLAND
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