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Protein Expression & Purification


Products >  Protein_Expression_and_Purification >  Protein_Learning_Center >  Tagged_Protein_Purification >  His-Tagged_Protein_Purification_Overview

His-Tagged Protein Purification Overview

Technologies

Takara Clontech offers four different technologies for his-tagged protein purification:

His Tag Miniprep

Magnetic Beads

His Tag Purification 101

Learn how to choose the best his-tagged protein purification method for your specific application. Mini spin columns are ideal for simple, rapid purification of small amounts of protein. The high capacity of His60 Ni Resin makes it an excellent choice for purifying large amounts of high-quality protein, while the Co2+ core of TALON resin allows it to deliver proteins of the highest purity. Both of these resins are available in several formats, including gravity columns, FPLC cartridges, and various sizes of bulk resin, as well as magnetic beads for microscale purification.

Comparison of His-Tagged Protein Purification Methods
  Purity Yield Binding capacity (mg protein/ml matrix) Start to finish (minutes) Ease of use
Capturem His-Tagged Purification Miniprep Kit **** 0.1 mg/mini spin column 100 mg/ml 5 min *****
His60 Ni **** 60 mg/ml resin 60 mg/ml 60–120 min ***
TALON ***** 20 mg/ml resin 20 mg/ml 60–120 min ***
Magnetic Beads ***** 15 mg/ml resin 15 mg/ml 60 min ****

 

What Makes His Tags Attractive?

Over 75% of researchers use his tags for purifying proteins, because they offer the following advantages:

  • Small size
  • Less likely to alter wild-type function/structure
  • Easy to clone
  • Effective when incorporated on either the N- or C-terminus
  • May be used to purify both native and denatured proteins

Advantages of Common His Tags
6xHis tag 6xHN tag HAT tag
  • Small tag (6 amino acids)
  • Works at C- or N-terminus
  • Small tag is less likely to interfere with protein function
  • Better for lower molecular weight proteins
  • Histidine-rich peptide (12 amino acids)
  • Works at C- or N-terminus
  • Long tag is less likely to internalize
  • Better solubility
  • More tag is exposed on the protein surface
  • Better for high molecular weight proteins
  • Naturally occurring sequence (18 amino acids)
  • Works at N-terminus
  • Longer tag is even less likely to internalize
  • Better solubility
  • More tag is exposed on the protein surface
  • Better for high molecular weight proteins
  • Proteins containing this tag are more stable

Choice of Metal Ions

Nickel- and cobalt-based resins offer a different balance of specificity vs. adsorption. The higher specificity of Co2+ provides greater purity, while the superior adsorption of Ni2+ provides a higher binding capacity.

Specificity vs. adsorbtion

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