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Immunofluorescence: polyclonal vs monoclonal antibodies and negative controls


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#1 bioche82

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Posted 11 September 2010 - 08:52 AM

Hi everyone,

I have a few questions about immunofluorescence.



Is it better to use a monoclonal or a polyclonal primary antibody?

Also, how do I verify for autofluorescence and background fluorescence?


Thank you very much.

#2 rkay447

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Posted 11 September 2010 - 02:39 PM

When it comes to IF, I've found that monoclonal versus polyclonal isn't a major consideration. It's more a matter of which antibody works. Polyclonal may give you more background but it's also more likely to stain your specific protein of interest. You should try both if you can. As for controls, you want to test your secondary antibody alone (no primary) to see what background this gives you. You may find you need to mess around with your fixation and blocking methods to optimize but then again, sometimes specific antibodies and cellular structures require a specific fixation. Centrosomes, for example, require methanol fixation. Autofluorescence would be what you see with no antibody staining, which in my experience is next to nothing but may depend on the cells or tissues being analyzed. If you do a google search you'll find ways to deal with or reduce autofluorescence if this is an issue. Generally, aldehyde fixatives are more prone to creating autofluorescence with gluteraldehyde being the worst. The best way to ensure specificity of the primary antibody is to preabsorb the antibody with either the protein of interest or the peptide to which the antibody was generated. Many companies offer the peptide as well as the antibody just for this purpose. You incubate the antibody with the peptide and then try to stain your samples. Any signal you see with this treatment is potentially nonspecific.

#3 bioche82

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Posted 12 September 2010 - 08:25 AM

Thank you very for your answer. I makes a lot of sense. However, what about replacing the primary antibody with the IgG from the species in which it was raised. Would it serve as a good control for the specificity of my antibody?

#4 bguyonnet

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Posted 23 September 2010 - 01:37 PM

Thank you very for your answer. I makes a lot of sense. However, what about replacing the primary antibody with the IgG from the species in which it was raised. Would it serve as a good control for the specificity of my antibody?

IgG is a good control but the blocked antibody seems to be the best one because with IgG you do not really know where they come from.. I tell that because I have problem with using IgG as a control for immunofluorescence. Now I am trying to block my first antibody.

#5 rkay447

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Posted 24 September 2010 - 10:15 AM

I agree that a "minimal" IgG control is perhaps the normal IgG from the same species (which you can buy from santa cruz, abcam, etc..) but you may run into problems because you never know where it came from or what that specific animal was exposed to. If you try normal IgG from different lots, you will likely see a wide variety of "background" staining. The ideal IgG control is pre-immune serum from the same animal that generated the specific antibody. I have only been able to obtain this when making my own antibody. You take some bleeds from the animal before injecting the antigen. This shows you what antibodies were present in that specific animal before making the antibody to your peptide. Any signal/staining you obtain with the pre-immune is non-specific. Again, I've never seen a commercial antibody offer this so your best control is the pre-absorbing.




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