Hi All,
I need to wash and recharge Ni-NTA column. Unfortunately, I do not have GuHCL now. Can we use 8M Urea instead of GuHCL? Is it also necessary to wash recharged beads with Urea+ 0.2 M acetic acid before I equilibrate with binding buffer? What is the function of acetic acid in stripping/washing Ni-NTA beads?
Protocol says it should be washed with 6M GuHCL+0.2M acetic acid first. Can we used only 8M Urea or Urea+ acetic acid?
Dear Abir,
as an alternative I can recommend using NaOH and EDTA for thorough stripping and regeneration. A suitable protocol you can find here:
https://cube-biotech.com/media/pdf/08/34/42/Washing_Regenerating_HisAffinityResins.pdf
The acetic acid is used to remove non-specifically bound nickel ions.
Best, Roland
Urea + acetic acid should work, although GuHCl has the added bonus of abolishing non-specific cation exchange interactions.
I was under the impression that using 0.2 M acetic acid was simply a cheap and easy way to lower pH and therefore protonate whatever His residue was still binding a Ni coordination site.
There is a booklet that used to be offered by Qiagen (I think it was called "the Qiaexpressionist") that included somewhere close to the end a thorough protocol for cleaning Ni-NTA-agarose resins. You might want to take a look at that for ideas; it includes washes with organic solvents and detergents to remove protein aggregates in addition to the usual stripping of Ni with EDTA, GuHCl/HAc, etc.
DEar Abir
as Roland suggest you can just strip away the metal and impurity with EDTA, wash with water and reload the metal (eg Nickel cloride)
generally after that the elution is complete, i regenerate the column in this way:
wash water milliQ (3CV)
metal stripping by EDTa 0,5M ph=8 (5CV)
wash with water (5-10CV)
bind metal - Nicl2 0,1M (5CV)
wash with water (5-10CV)
equilibrate with binding buffer or store with water/etanol 20%
ciao
Manuele
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