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Can't see the immunoprecipitated protein

Can't see the immunoprecipitated protein - Immunoprecipitation Forum

Can't see the immunoprecipitated protein - IP or Immunoprecipitation Forum.


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  #1  
Old 02-20-2011, 06:54 PM
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Cool Can't see the immunoprecipitated protein



Hi

I'm new to the IP world, so your help is much appreciated!

I'm trying to IP protein Y and blot for protein X to see if the two proteins form a complex. I use rabbit IgG as my negative control, since the antibody i used to precipitate protein Y is rabbit. When I blot for protein X, I see the specific bands for the protein present in the IP sample, and absent in the negative control sample - so far so good. However, when I blot for protein Y (using a mouse antibody) as a loading control, I can't see any protein Y....

Any idea why?

Thanks for your help in advance!

K
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Old 02-21-2011, 03:28 AM
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Default Re: Can't see the immunoprecipitated protein

So your isolation of X works, neg control is neg., and positive control shows X.

But the positive control doesn't show Y, which is the actual captured antigen. Funny I'd suspect that the capture antibody-antigen Y complex is much stronger than Y-X heterodimer (??) and whatever your eluting with is only strong enough to disrupt Y-X (thus X is present for your experiment). Thus, possibly leaving antibody-Y complex on beads or whatever you are using.

So try stronger elution buffers, and possibly a carrier protein or denaturant (SDS or urea) to protect from protein degradation.

If you can't rule out degradation yet, check for half-life estimates for the X protein. this tool works for me ---> [Only registered users see links. ]
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Old 02-23-2011, 01:37 AM
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Default Re: Can't see the immunoprecipitated protein

thank you so very much danfive! what you said make sense!
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Old 05-18-2011, 10:20 PM
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Smile Re: Can't see the immunoprecipitated protein

There are many potential problems for IP. From your description I'd suggest that you need to rule out the possibility of dissociation of X-Y proteins in your sample buffer. For IP it's always better to use native protein extract than the denatured one. There are many buffers/kits you can use. You can try a protein extraction kit from Invent biotechnologies which is native and super-fast.

If you know where you X-Y protein complex is located you can isolate the fractions that contain majority of the complex (Cytosol vs. nuclear fraction) and increase the efficiency of IP.
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