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Stripped and recharged Ni-NTA beads but still getting impurities only with Ni-NTA beads

Phelan, Paul J. via methods%40net.bio.net (by Paul.Phelan from tufts.edu)
Fri Aug 4 13:47:24 EST 2017


I have had protein stick to Ni-NTA agarose that did not come off even when I stripped the nickel with EDTA.
In this case, you can use a strong cleaning with 0.5 M NaOH (10 column volumes, followed immediately with at least 10 column volumes H2O, do not let NaOH sit on the resin) to remove denatured protein.  This was the only treatment that would remove the protein stuck to the resin in my case.  You can also do a separate wash with 10 column volumes 70% ethanol, if you think there is protein bound hydrophobically to the resin.

Paul Phelan
Tufts University School of Medicine
Boston

________________________________________
From: methods-bounces from oat.bio.indiana.edu [methods-bounces from oat.bio.indiana.edu] on behalf of Sudheer Sangeetham [sudheer.pbm07 from gmail.com]
Sent: Friday, August 04, 2017 6:18 AM
To: Methods from magpie.bio.indiana.edu; methods from oat.bio.indiana.edu
Subject: Stripped and recharged Ni-NTA beads but still getting impurities only with Ni-NTA beads

Hi

*  Strip of nickel ions by washing with 10 bed volumes of 100 mM EDTA, pH
8.

* Wash resin with 10 bed volumes of de-ionized water.

* Charge resin with 2 bed volumes of 100 mM NiCl2 metal ion aqueous
solution.

* Wash resin with 10 bed volumes of de-ionized water to remove unbound
metal ions.

* Stored the resin in 30% etanol.

I loaded only recharged bead on the gel to check I could see nothing on the
sds-page or not, but I could see faint or visible proteins on sds page due
to protein and Ni didnt strip off completely.

please let me any solution for it.


Thank you


Regards
Sudheer
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