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Support >  Applications >  Tagged_Protein_Purification >  Ni-NTA_Resin_vs._Talon

His-Tag Purification Resins- TALON Cobalt- Highest Purity

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.

Obtain better purification of his-tagged proteins with cobalt resin than with Ni-NTA. Purify his-tagged protein at milder pH with TALON resin than with Ni-NTA.

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.
Preserve biological activity of his-tagged proteins with TALON resin. TALON resin purifies his-tagged proteins under denaturing conditions.

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)

Reduced sulfhydryl (-SH) groups are important to the biological activity and structure of some intracellular proteins, and BME is often used to preserve these reduced -SH groups. TALON resin provides higher yields of his-tagged proteins that contain reduced -SH groups in the presence of BME compared to Ni-NTA resin.


Obtain higher yields of BME-treated, his-tagged protein with TALON cobalt resin than with Ni-NTA.

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:

  1. 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.
TALON cobalt resin is highly selective for his-tagged proteins.
  1. 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.

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

  1. Chaga, G. S., et al. (1999) Biotechnol. Appl. Biochem. 29(1):19—24.
  2. Porath, J. (1992) Protein Express. Purif. 3(4):263—281.
  3. Yang, T., et al. (1997) Amer. Biotechnol. Lab., pp. 12—14.

 
 
Products
Cat. # Product Package Size Price License # of Units Select
635501 TALON® Metal Affinity Resin 10 mL $95.00 License Statements
635502 TALON® Metal Affinity Resin 25 mL $212.00 License Statements
635503 TALON® Metal Affinity Resin 100 mL $718.00 License Statements
635606 TALON® 2 ml Disposable Gravity Column 50 Columns $95.00  
635504 TALON® Metal Affinity Resin 250 mL $1,617.00 License Statements
635652 TALON® Metal Affinity Resin 2 x 250 mL $2,266.00 License Statements
635653 TALON® Metal Affinity Resin 4 x 250 mL $4,299.00 License Statements
 

 

 

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