Knight:Purification of His-tagged proteins/Denaturing

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==Overview==
==Overview==
"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." <cite>QiagenNTAManual</cite>
"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." <cite>QiagenNTAManual</cite>
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 +
==Materials==
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 +
===Lysis and column equilibration buffer (Qiagen buffer B)===
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*8 M urea
 +
*100 mM NaH<sub>2</sub>PO<sub>4</sub>
 +
*10 mM Tris Cl
 +
*pH 8.0
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 +
===Wash buffer (Qiagen buffer C)===
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*8 M urea
 +
*100 mM NaH<sub>2</sub>PO<sub>4</sub>
 +
*10 mM Tris Cl
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*pH 6.3
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===Elution buffer (Qiagen buffer E)===
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*8 M urea
 +
*100 mM NaH<sub>2</sub>PO<sub>4</sub>
 +
*10 mM Tris Cl
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*pH 4.5
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==Notes==
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*Sauer lab uses a Qiagen Ni NTA resin but we have an old kit with spin columns.
==Safety==
==Safety==
-
*Buffer A has guanidine hydrochloride in it.
+
*Some buffers may have guanidine hydrochloride in it.
==References==
==References==

Revision as of 16:47, 11 July 2006

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.

Contents

Overview

"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]

Materials

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

Notes

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

Safety

  • Some buffers may have guanidine hydrochloride in it.

References

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