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Co-IP problem


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#1 aquablue

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Posted 10 May 2009 - 09:55 PM

Hi everyone. I have a question regarding Co-IP and I would greatly appreciate any help.

So I IP-ed mammalian cell lysate with a mouse antibody and I blotted with a different Ab that is also mouse in origin. After the Western, there does seem to be a band in the size I want. However, my IgG heavy chain band migrated a little faster than the control lane. Also its signal is weaker than that in the control lane. In addition, this IgG band has a bent 'smiley' shape, whereas the IgG bands in other lanes look completely straight.

Do you reckon there is something wrong with my IP, or this is simply because there is more protein in this particular IP (my wild guess...)?

Since biochemistry is not my strength, i would greatly appreciate any explanations/suggestions from the forum-ers. Thanks in advance!

#2 mikew

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Posted 11 May 2009 - 12:09 PM

Hi,

#1. Don't do westerns of an IP with the same species antibody. This leads to artifacts and non-specific bands.
#2. Your heavy chain migrated faster in the IP lane probably due to a different amount of antibody being in that lane. Signal is weaker in control lane due to different amount of antibody.
#3. As for the smiley face, I don't really know, make new buffers and maybe run your gel more slowly.
Smiley bands usually result from heat.




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