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only monomer immunoprecipitates


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#1 neurophdstudent

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Posted 13 July 2009 - 01:47 PM

I am trying to immunoprecipitate alpha-synuclein, which is a peptide that aggregates. I am only able to IP the monomer. I would like to get the aggregates as well. The antibody I am using binds the aggregates of alpha-synuclein, as shown by Western blot, so the antibody has the capacity of binding these aggregates.

I am using Protein A/G Plus Agarose, and the antibody is rabbit polyclonal. I incubate the antibody in the column for 1 hr, incubate crosslinker for 1 hr, and incubate the homogenate in the column overnight. These are done at 4C while rocking.

I am not sure if I should optimize this protocol or if the problem is actually caused by something else.

Please advise.

Thank you.

#2 miBunny

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Posted 13 July 2009 - 05:26 PM

This sounds similar to a Co-IP style procedure. The big things to consider are the salt and detergent concentrations in your lysis and IP buffers. You don't want too much detergent and/or salts or you favor the disaggregation of your aggregates. If there are any published protocol for this protein (or something similar), thats always a place to start. Also, you may want to avoid vortexing your samples during the IP procedure as that may manually disrupt the aggregates.
Good luck! These things can be a pain to work out!

#3 neurophdstudent

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Posted 14 July 2009 - 11:16 AM

Thank you so much for your suggestions, miBunny. I have checked prior articles, and it has been done before using similar protocol. Also, we use the same protocol to IP another peptide, amyloid beta, and we can get the aggregates.

Your ideas are brilliant and as I think every time I attempt science, I wish the answer could just slap me in the face..

Thanks again.




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