Re: triple labeling with antibodies
Posted by
lechristophe on
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Dear Shalin,
I don't know about horse secondary antibodies, but I think you shouldn't use goat primary and goat secondary on the same sample, even if they're highly cross-adsorned. We routinely do triple labeling with goat/mouse/rabbit primary antibodies, using donkey secondary (donkey anti-goat, anti-mouse and anti-rabbit). Alternatively, we also use the rat/mouse/rabbit and chicken/mouse/rabbit combination with goat secondary antibodies.
As regards your spectral choice, what is the ratinale behind choosing 514 as the first color ? Is there a reason you don't want to use 488 ?
Christophe Leterrier
On Dec 13, 2007 8:58 AM, Shalin Mehta <
[hidden email]> wrote:
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Dear all,
I am getting out of my depth about triple labeling with antibodies.
We have three molecules that we want to label and see simultaneously
with AF514, AF555 and AF633. We have decided to use goat, mouse and
rabbit antibodies for three antigens. Now, it seems pretty
straightforward to use e.g. horse anti-goat, horse anti-mouse and horse
anti-rabbit secondary antibodies (I didn't know about the issue of
cross reactivity.) I noticed on Invitrogen website that they offer
'highly adsorbed' secondary antibodies (goat anti-mouse and goat
anti-rabbit) for multiple labeling experiments. Does it mean that we
should be safe using, e.g., horse anti-goat, highly adsorbed goat
anti-rabbit and highly adsorbed goat anti-mouse? I am not sure what
cross reactivity means and what logic dictates choice of secondary
antibodies.
Any explanation will be helpful.
Thanks
Shalin
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Shalin Mehta
Graduate Student in Bioengineering, NUS
mobile: +65-90694182
blog:
shalin.wordpress.com
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