how do you inactivate His-tag affinity beads?? - (Apr/16/2014 )
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!
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.
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.
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.
Great! Thanks for your help!