His-Tagged Protein Purification: Obtain Highest Purity with Cobalt Resin
Reduced copurification of impurities
| Works under native and denaturing conditions | Compatible with BME
| Resists metal leakage Why Use TALON Cobalt Resin for His Tag Purification?
TALON His-Tag Purification Resin lets you prepare exceptionally pure his-tagged proteins from bacterial, mammalian, yeast, and baculovirus-infected cells, under native or denaturing conditions. TALON is an immobilized metal affinity chromatography (IMAC) resin charged with cobalt, which binds to his-tagged proteins with higher specificity than nickel-charged resins. As a result, TALON resin delivers his-tagged proteins of the highest purity.
TALON Cobalt Resin Beats Ni-NTA Resin
The cobalt ions in TALON are more selective for histidine tags than nickel ions are. This means that using Ni-NTA resins often results in copurification of contaminants. Another advantage of TALON resin is that you can elute your his-tagged protein at a mild pH or lower imidazole concentration.
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Figure 1. 6xHN-AcGFP1 was purified from Sf21 cells using TALON (left) or Ni-NTA (right). The chromatogram for each column is shown. The absorbance at 475 nm indicates the amount of target protein (AcGFP1) present in each fraction. |
Figure 2. 6xHis-tagged protein (prepro-alpha-factor) was loaded onto TALON and Ni-NTA columns, eluted by stepwise pH, and analyzed by SDS-PAGE. The protein was eluted from TALON resin at pH = 6.3 and from Ni-NTA resin at pH = 4.5. |
Purify His-Tagged Proteins Under Native or Denaturing Conditions with TALON Resin
TALON Resin retains its protein binding specificity and yield under a variety of purification conditions. TALON resin is stable under native purification conditions and under denaturing conditions, e.g. purifying his-tagged proteins with urea.
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Figure 3. The fluorescent signal of green fluorescent protein (GFPuv) was completely enriched by TALON resin. 6xHis-tagged GFPuv was extracted from fresh cells and purified using TALON cobalt resin. Panel A. GFP elution profile. Panel B. SDS-PAGE analysis. |
Figure 4. Purification of 6xHis-tagged GFPuv under denaturing conditions. The fusion protein was purified in 8 M urea using TALON cobalt resin. M=molecular weight markers. |
TALON Resin is Compatible with Beta-Mercaptoethanol (BME)
BME helps to preserve the reduced sulfhydryl (-SH) groups that are important for the biological activity and structure of some intracellular proteins. TALON provides higher yields of his-tagged proteins in the presence of BME compared to Ni-NTA resin. |
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Figure 5. N-terminal 6xHis-tagged DHFR was expressed and purified under native conditions. Protein concentrations
were determined by Bradford assay. Yields are expressed as a percentage of total protein in the cell lysate. |
TALON Resin: Cobalt is the Key to His-Tagged Protein Purification
TALON resin is based on the cobalt ion, which is highly selective for his-tagged proteins (1-3). This makes TALON resin a better choice than Ni-NTA resin:
- TALON resin has better specificity for his-tagged proteins than nickel-NTA. In TALON, only proteins containing adjacent histidines or specially positioned, neighboring histidines are able to bind. By comparison, the spatial requirements for Ni-NTA resin are less strict—these resins have a much higher affinity for randomly positioned (i.e., non-his tag) histidines. As a result, TALON resin binds more specifically to polyhistidine-tagged proteins and provides higher purity.
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- TALON resin has low metal ion leakage. Cobalt resin has a very uniform three-dimensional structure in which cobalt is bound very tightly. Nickel-based resins are less homogeneous in structure because nickel ions can form two different coordination complexes, one of has a planar (flat) structure that does not bind the nickel ions tightly. Thus, nickel ions can leach from the resin.
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Why is metal ion leakage bad?
- Metal leakage reduces the number of reactive sites available for protein binding, and therefore reduces the yield of purified protein.
- Free metal ions have a detrimental effect on protein activity.
- Metal ions can precipitate proteins by forming salt bridges, can be toxic to cells and tissues, and can damage purified protein because of its nucleophilic properties.
We Also Recommend
- His-tag Detection Antibodies
Use highly sensitive antibodies to detect his-tagged recombinant proteins in Western blot, ELISA, and immunocytohistochemical assays.
- ProteoGuard Protease Inhibitor Cocktail
Use this protease inhibitor cocktail to suppress proteolysis in all your cell lysates. We recommend using ProteoGuard with all our protein purification products.
References
- Chaga, G. S., et al. (1999) Biotechnol. Appl. Biochem. 29(1):19—24.
- Porath, J. (1992) Protein Express. Purif. 3(4):263—281.
- Yang, T., et al. (1997) Amer. Biotechnol. Lab., pp. 12—14.
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