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stripped Ni column turn brown yellow

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#1 genz



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Posted 13 May 2009 - 07:59 PM

I obtain a large amount of small membrane protein (13 KDa) expressed in E coli. It's solubilised in 1% TX. It bind extremely well in Ni colum (GE lifescience brand).
My elution buffer is 20 mM Phosphate pH 7.4, 150 mM NaCl, 0.05% TX, 300 mM Imidazole.
After eluting the protein, the top part of the column turns brown. I stripped it with 50 mM EDTA, the green Ni is gone, but the top part still remains brown. I tried running ammonium persulphate to oxidise it, but the top part of the column is still brown.
Can anyone help!?! :D

#2 mdfenko


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Posted 15 May 2009 - 11:07 AM

the brown may be aggregated and precipitated proteins (and/or other contaminants or a change in the valence of the nickel). try running naoh through the column then regenerate or just follow the regeneration instructions that came with the column.

you may want to determine if the brown material has an effect on the running of the column or on the product. if not then you may be able to ignore it.
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