Re: triple labeling with antibodies

Posted by rjpalmer on
URL: http://confocal-microscopy-list.275.s1.nabble.com/triple-labeling-with-antibodies-tp591231p591246.html

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No I'm even more skeptical.  I'd recommend that, in the absence of sophisticated excitation control together with good detection separation (Meta or SP), you try using the GFP and 514 together before making a big commitment in that direction.

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Thanks for comments. We also hope to see GFP with these, so AF488 is not used.
By the way, I actually was also asking a novice explanation of what is involved in high adsorption. What are antibodies adsrobed on (which is the process of accumulating some solute on solid surface that attracts it)? How does it reduce cross-reactivity?

Best
Shalin
 

On Dec 13, 2007 6:24 PM, Farid Jalali <[hidden email]> wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello Shalin,
I agree completely with Christophe's' comments. Something that our own lab has started to test for tri-labeling is the Zenon kit/product line from Invitrogen. You can generate your own primary conjugated antibodies quite easily. We often have primary antibodies from the same host that we would like to use together and this product is a very efficient way of doing this without having to search for an antibody from a different host that we would have to test. Labs with stronger ties to chemistry can actually find ways of doing this without this kit. What we have found is that the product most certainly works, but produces a slightly fainter signal.

Good Luck
Farid

On Dec 13, 2007 4:13 AM, Christophe Leterrier <[hidden email] > wrote:

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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:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 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
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Farid Jalali MSc
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Applied Molecular Oncology

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Shalin Mehta
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blog: shalin.wordpress.com
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