sorry in advance for maybe a "lame" question but I would like to know wheter this costaining primary/secondary antibodies combination will function correctly:
anti-Integrin Alpha 2 (mouse, mono, IgG2A) + goat anti mouse AF 568 IgG2A
anti-Integrin Alpha 5 (goat, poly, IgG) + donkey anti goat AF 488 IgG
Obviously I want to stain two different Alpha-Integrins. The thing is...Can I put these secondary antibodies together in one costaining experiment? Will the donkey anti-goat bind to goat anti-mouse? Is the isotype enough to distinguish the specificity?
Thanks in advance
Wed Aug 17, 2011 9:01 am
Joined: Oct 11, 2004
Post subject: Hi Anthos
Not a lame Q at all...
donkey anti goat AF 488 IgG will bind to goat anti mouse AF 568 IgG2A
as well as your goat primary, imho.
You could use a rabbit anti Ms IgG 568...
I may be wrong.
Wed Aug 17, 2011 1:07 pm
Joined: Mar 20, 2008
1. mouse primary
2. goat primary
3. donkey secondary binds to goat
4. goat secondary binds to mouse
I think you can mix the primaries in one step, but the secondaries have to be applied successive.
Wed Aug 17, 2011 6:39 pm
Joined: Aug 17, 2011
Post subject: thanks
Thanks for the answers, your thinking is similar to mine. If there is another opinion somewhere, do not hesitate to express it.
Fri Aug 19, 2011 5:17 am
Joined: Oct 11, 2004
Post subject: hmmmm also lol
I reckon that the secondaries will x-react even if applied sequentially.
Do not use GOAT anti mouse....because it will link up with donkey anti GOAT?
Anyway..test it out.
I am interested why you use AF568.....why not AF594?
It seems to me that the emissions of AF488 and AF594 are further apart on the spectrum, so better separation?
So, why do so many people use AF 568?
Please show me that I am wrong.
Fri Aug 19, 2011 7:58 pm
Joined: Jul 07, 2008
Carl is right, also if you incubate the two secondaries separately you will end up with aspecific double staining. Which means that you have to use secondaries from one host (donkey-anti mouse IgG2a together with donkey-anti-goat). You don´t have to use an anti-IgG2a , every anti-IgG will detect it. In this way you can incubate both primaries and in a second step both secondaries.
Generally I think it is a good idea to get all your secondaries from one host (preferably donkey) because you then never run into these aspecific double stainings. Why donkey? Because most primary antibodies are mouse, rabbit or goat/sheep.
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