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Using 2˚ antibody as a isotype control?


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#1 Namako

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Posted 03 May 2011 - 06:06 PM

Is it okay to use a secondary antibody as an isotype control for a primary conjugated antibody? I am wanting to use a rat anti-mouse secondary antibody as the isotype control for a rat anti-mouse primary conjugated antibody (they have the same flurophore and are the same isotype). I am worried that the secondary antibody could give me more background than a normal isotype control. (Maybe it would have a higher likelihood of binding to any Fc receptors on my mouse cells, since the secondary antibody is anti-mouse?) I have heard that one can use an irrelevant antibody for a particular tissue type as an isotype control, but I don't have any. It would be great if I could feasibly use the secondary antibody instead, but I don't want to have a higher background than a designated isotype control, as I am looking for a stem cell population that isn't particularly abundant.

Anyone have any knowledge about this? Thanks! :)

#2 knuf

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Posted 04 May 2011 - 06:16 AM

I don't think using a secondary antibody as your control is a very good idea. If you're really at a loss for a proper isotype control, then you could use non-immunized mouse serum.

#3 Namako

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Posted 04 May 2011 - 01:38 PM

Great, thank you. So just to make sure I'm understanding, an isotype control antibody is produced in non-immunized animals, correct?

#4 knuf

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Posted 05 May 2011 - 05:59 AM

Well, no...and isotype control antibody would be an antibody that is the same isotype as your antibody of interest...so if the antibody you are using is mouse IgG, then the isotype is mouse IgG and the best control would be another antibody that is mouse IgG but that is not expressed in your sample--like a neuron-specific antibody in muscle tissue or something like that. A second less good control is mouse serum from a non-immunized mouse which is going to also have IgG, but not as much and not purified.

#5 Namako

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Posted 25 May 2011 - 04:43 PM

Great, thank you!




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