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how do you inactivate His-tag affinity beads??


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#1 rockstar

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Posted 16 April 2014 - 08:10 PM

Hi all,

 

I'm going to do an IP experiment soon. I will have a duplicate of the sample that will be either IPed with His-SELECT affinity gels or IPed with inactivated gels (for control). My question is what's the best way to inactivate the beads before the IP experiment. I can think of 2 options: boil them for 10minutes or treat them with high concentration of imidazole. Anyone has any suggestion?

 

Thanks in advance!



#2 dimensionsbio

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Posted 18 April 2014 - 03:30 AM

Hi Rockstar.  I am not really sure what you mean by inactivate the bead.  If you want treat the beads so that they wont specifically bind to His-tagged protein to look at nonspecific binding to the agarose, then just remove the Ni2+.  Use EDTA to do this.



#3 rockstar

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Posted 21 April 2014 - 07:46 PM

Hi dimensionsbio, thanks for your reply.

 

What I want to do is to treat the beads so they won't be able to bind to His-tagged protein anymore, basically don't want the beads to be able to bind to anything anymore. Maybe it seems odd, but what I want to do is to have this "inactive" beads as a negative control. I need to still incubate the sample with this "inactive" beads to make sure that the volume between this negative control sample and the real sample is the same.



#4 phage434

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Posted 22 April 2014 - 05:04 AM

If you wash your beads in high concentration EDTA, it will remove the nickel (or cobalt) on the beads, making them inactive. You should see a color change.

Wash a second time  (or more) in your buffer of choice.



#5 rockstar

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Posted 27 April 2014 - 07:17 PM

Great! Thanks for your help!






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