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Antibody vs Antigen concentration effect


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#1 ghostrider

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Posted 29 June 2011 - 04:32 AM

Someone once described to me that if there are too many non-specific bands when performing Western blot that a solution is to load more lysate? If this is true would one achieve the same effect if the lysate amount is held constant and the antibody concentration titrated? I have been trying to track down a literature article describing this. Hoping someone can shed some light.

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#2 bob1

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Posted 29 June 2011 - 04:49 PM

Typically it is the antibody concentration that is titrated. I don't think that adding more protein will have the same effect if your specific signal is already strong. If your signal is very faint you can try running more protein per lane.

#3 ghostrider

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Posted 30 June 2011 - 05:48 AM

Typically it is the antibody concentration that is titrated. I don't think that adding more protein will have the same effect if your specific signal is already strong. If your signal is very faint you can try running more protein per lane.


I will explain in more detail. Basically what I am observing is a significant amount of non-specific bands appearing without the band of interest. The amount of lysate loaded onto the gel was 50ug which in our experience should be sufficient. Trying to detect a moderately expressed transcription factor. What was told to me was that if I load more lysate > 100ug that our band of interest will appear and the non-specific bands will disappear. Basically, playing the s/n game. In my experience I have never seen this inversion happen. My guess this only applies to extremely poor, low affinity antibodies. Any insight and/or literature ref explaining this phenomenon would be greatly appreciated.

#4 bob1

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Posted 30 June 2011 - 05:52 PM

I've never heard of that working before either - usually if you can see non-specific bands they will be there when the specific band is there too.

I suppose it could work a bit like PCR - if there is no specific target around, the primers (or antibody) might bind to any sequence and give you some signal.




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