Immunoprecipitation

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rachawilson
rachawilson's picture
Immunoprecipitation

Hello all,

I have been working with immunoprecipitations for a short while and I always use a control with IP extract and no antibody.  What I have been hearing is that a more proper control for IPs is the use of a control IGG antibody is this correct? If so can someone explain in detail the reasoning behind this.  Lets say I am incubating my extract with a proteinX antibody that is anti-mouse, and then on the western blot I probe for proteinZ that is also anti-mouse....does the control IGG need to be anti-mouse?

Would my negative control for this co-IP be (IP extract + control IGG anti mouse antibody)?

Please help. Thanks.

Chin Fen Teo
Chin Fen Teo's picture
 Hi rachawilson,

 Hi rachawilson,

The control IgG should be normal mouse IgG, not anti-mouse IgG. The idea behind is to make sure the specificity of your IP result.

In short, when you perform protein X WB with your IP sample, you should not see any thing with your normal IgG IP around the region where protein X localize, but a specific bands with protein X antibody IP.

The choice of the normal IgG for control IP always refer to the species of antibody for IP, but irrelevant to the species of antibody for WB.

Hope this helps.

rachawilson
rachawilson's picture
So the control mouse IgG

So the control mouse IgG should be placed at the same amount (ug) as my antibody of interest? 

Also, is there a way you can link me to a site with control mouse IGG from a company?

Chin Fen Teo
Chin Fen Teo's picture
 I usually use the same

 I usually use the same amount of Ab.

This is the link where I got my normal mouse IgG.

Good luck.

rachawilson
rachawilson's picture
Much appreciated. Thank you.

Much appreciated. Thank you.