questions about IP , - (Jun/08/2007 )

it is about co IP. I did IP with one mono Ab whose second antibody is antimouse. then I did western on this IP with this antibody, second antibody is also antimouse. then I get hugh black thing, I think it is because my western second antibody picks up the antibody I used for IP. What should I do to get rid of this and see my real band?

Thanks

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Use different primary Antibodies (different species) in your WB. What you saw is the heavy chane of your IP antibodies.
Also, there is a wonderful kit out there for IP/CO-IP if you don't have that much of choices in antibodies. Its called profound Co-IP kit.


thanks

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Thanks . I checked online, PIERCE has this one, but quite costly 40IP for 263 pounds. Which one you've used? and how much is it?

Thanks again.

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Thanks anwar-mt. Is it a general rule that if the antibody for use in IP and western are same species. the western will pick up heavy chain always, so marked the real band. or it is also depending on the antibody, some of them might still see the real band?

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is it a very stupid question? so nobody interested?

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Hi Cathy!!!
It is a common problem. What you can do not to detect the heavy chain of ab is to incubate your 1st ab (let's say mouse monoclonal) with 2ndary ab in the tube and afterwards add mouse Serum which will bind to the 2ndary antibody which is not bound to the 1st antibody and then when you incubate with your blot there is no free 2ndary ab which can bind to the heavy chain.
You might read this paper where everything is described in detail.

Suppression of Irrevelant Signals In Immunoblots By Preconjugation of Primary Antibodies
Joachim Langstein and Herbert Schwarz
BioTechniques Vol. 23, No. 6: pp 1006-1010 (Dec 1997)



Hope this will help,
Karmen

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