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unspecific binding

unspecific binding - ChIP Chromatin Immunoprecipitation Forum

unspecific binding - Chromatin Immunoprecipitation Forum. Discuss, chat about, and post questions about the ChIP assay, in the ChIP forum. Includes troubleshooting, protocols, buffer preparation, and more.


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  #1  
Old 11-26-2008, 12:27 PM
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Default unspecific binding



Hello.
Can anyone tell me if there are sequences that can bind unspecific to Protein A Agarose more easy then others? Or is it just a matter of size?
Thank you very much.
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Old 11-26-2008, 08:05 PM
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Default Re: unspecific binding

Hello,
there may be some sequences that do it more often that others. Not sure about size.

Have you tried non-specific competitors? Using BSA? other methods to block non-specific binding such as harsher conditions?

cheers
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Old 11-27-2008, 11:45 AM
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Default Re: unspecific binding

My Protein A Agarose is pre-blocked with salmon sperm DNA, and before I add the immunoprecipitating AB, I pre-clear the eluates with Protein A Agarose, that I discard afterwards.
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Old 12-03-2008, 03:05 PM
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Default Re: unspecific binding

hmm any progress on this I may also conduct Chromatin Immunoprecipitation soon... maybe has to do with the Protein A?
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Old 05-08-2009, 09:51 AM
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Default Re: unspecific binding

Did you check the sheared DNA on gel? what's the size distribution?

Also,You generally have much less of these bacgkround problems if you use magnetic beads instead of agarose.
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Old 05-08-2009, 09:52 AM
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Default Re: unspecific binding

Did you check the sheared DNA on gel? what's the size distribution?

Also,You generally have much less of these bacgkround problems if you use magnetic beads instead of agarose.
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