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Ni-NTA column turns yellow colour
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sudheer sangeetham
Nov 15, 2012, 10:27:19 AM11/15/12
to Met...@magpie.bio.indiana.edu
Hi everyone
After elute the protein from Ni-NTA beads I washed the beads with 0.5M NaOH
to get rid off imadozole and traces of protein on the bead, but
unfortunately the column turns yellow colour? My recombinant protein which
fusioned to m-cherry, even after elution the column looks light pink in
colour. could anyone explain why it has happened and how I could get back
Ni-NTA beads to normal light blue color.
Thank you in advance
--
Sudheer Babu.S
Research Fellow
Institute of Biochemistry
Biological Research Center
Szeged,Hungary.
After elute the protein from Ni-NTA beads I washed the beads with 0.5M NaOH
to get rid off imadozole and traces of protein on the bead, but
unfortunately the column turns yellow colour? My recombinant protein which
fusioned to m-cherry, even after elution the column looks light pink in
colour. could anyone explain why it has happened and how I could get back
Ni-NTA beads to normal light blue color.
Thank you in advance
--
Sudheer Babu.S
Research Fellow
Institute of Biochemistry
Biological Research Center
Szeged,Hungary.
WS
Nov 15, 2012, 7:20:18 PM11/15/12
to Met...@magpie.bio.indiana.edu
Hi Sudheer,
seems your column became oxidized, possibly 0.5M NaOH was a little too harsh? (broke the complex and precipitated the Ni as "hydroxide" which then got oxidized by oxygen dissolved in the solutions?)
Anyway, flush your column with a few ml of 0.1...0.5M EDTA (removes Nickel), water (removes EDTA) and then re-charge it with a few ml of 100mM Nickel chloride or sulfate, then again water and then your buffer of choice.
Please remember Nickel salts have some carcinogenic and allergenic potential and that Ni is a heavy metal that likes to be disposed of properly.
If the colored stuff does not disappear with EDTA initially, try water, 100mM HCl, followed by water, first.
HTH
Wo
seems your column became oxidized, possibly 0.5M NaOH was a little too harsh? (broke the complex and precipitated the Ni as "hydroxide" which then got oxidized by oxygen dissolved in the solutions?)
Anyway, flush your column with a few ml of 0.1...0.5M EDTA (removes Nickel), water (removes EDTA) and then re-charge it with a few ml of 100mM Nickel chloride or sulfate, then again water and then your buffer of choice.
Please remember Nickel salts have some carcinogenic and allergenic potential and that Ni is a heavy metal that likes to be disposed of properly.
If the colored stuff does not disappear with EDTA initially, try water, 100mM HCl, followed by water, first.
HTH
Wo
WS
Nov 15, 2012, 7:20:18 PM11/15/12
to bionet.molbio....@googlegroups.com, Met...@magpie.bio.indiana.edu
Hi Sudheer,
seems your column became oxidized, possibly 0.5M NaOH was a little too harsh? (broke the complex and precipitated the Ni as "hydroxide" which then got oxidized by oxygen dissolved in the solutions?)
Anyway, flush your column with a few ml of 0.1...0.5M EDTA (removes Nickel), water (removes EDTA) and then re-charge it with a few ml of 100mM Nickel chloride or sulfate, then again water and then your buffer of choice.
Please remember Nickel salts have some carcinogenic and allergenic potential and that Ni is a heavy metal that likes to be disposed of properly.
If the colored stuff does not disappear with EDTA initially, try water, 100mM HCl, followed by water, first.
HTH
Wo
On Thursday, November 15, 2012 11:27:19 PM UTC+8, sudheer sangeetham wrote:
seems your column became oxidized, possibly 0.5M NaOH was a little too harsh? (broke the complex and precipitated the Ni as "hydroxide" which then got oxidized by oxygen dissolved in the solutions?)
Anyway, flush your column with a few ml of 0.1...0.5M EDTA (removes Nickel), water (removes EDTA) and then re-charge it with a few ml of 100mM Nickel chloride or sulfate, then again water and then your buffer of choice.
Please remember Nickel salts have some carcinogenic and allergenic potential and that Ni is a heavy metal that likes to be disposed of properly.
If the colored stuff does not disappear with EDTA initially, try water, 100mM HCl, followed by water, first.
HTH
Wo
On Thursday, November 15, 2012 11:27:19 PM UTC+8, sudheer sangeetham wrote:
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