Knight:Purification of His-tagged proteins/Denaturing

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In progress! If you want to purify His-tagged proteins, I recommend using Sauer:Purification of His-tagged proteins not this protocol!

This page is really just for notes purposes for Reshma.



"The high affinity of the Ni-NTA resins for 6xHis-tagged proteins or peptides is due to both the specificity of the interaction between histidine residues and immobilized nickel ions and to the strength with which these ions are held to the NTA resin." [1]


Lysis and column equilibration buffer (Qiagen buffer B)

  • 8 M urea
  • 100 mM NaH2PO4
  • 10 mM Tris Cl
  • pH 8.0

Wash buffer (Qiagen buffer C)

  • 8 M urea
  • 100 mM NaH2PO4
  • 10 mM Tris Cl
  • pH 6.3

Elution buffer (Qiagen buffer E)

  • 8 M urea
  • 100 mM NaH2PO4
  • 10 mM Tris Cl
  • pH 4.5


  • Solid urea to make up an 8M solution takes up a lot of volume so be conservative on how much H 2O you start with (maybe 50% of final volume)
  • When initially made up the solution had a pH of 5.88.
  • The lysis buffer requires many drops of 5M NaOH (tens) to bring to pH 8.0.
  • The elution buffer only needs a little (few drops) of 1M HCl to bring to pH 4.5.


  • Sauer lab uses a Qiagen Ni NTA resin but we have an old kit with spin columns.


  • Some buffers may have guanidine hydrochloride in it.


  1. Qiagen Ni NTA Spin Kit manual [QiagenNTAManual]
  2. Sauer:Purification of His-tagged proteins/Denaturing prep


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