His-tagged protein purification
High-throughput, micro-scale purification of his-tagged proteins—TALON Magnetic Beads
Combine the advantages of our highly selective TALON chemistry with magnetic bead separation. Magnetic particles in the beads facilitate quick and easy separation of microscale quantities of protein when placed on a magnetic separator. The beads, which are precharged with Co2+, have a higher specificity for his-tagged proteins than nickel-based resins. Co2+ is bound to the beads using TALON's unique tetradentate metal chelator, which binds cobalt at four sites, virtually eliminating metal leakage during purification.
Combine the advantages of our highly selective TALON chemistry with magnetic bead separation. Magnetic particles in the beads facilitate quick and easy separation of microscale quantities of protein when placed on a magnetic separator. The beads, which are precharged with Co2+, have a higher specificity for his-tagged proteins than nickel-based resins. Co2+ is bound to the beads using TALON’s unique tetradentate metal chelator, which binds cobalt at four sites, virtually eliminating metal leakage during purification.
Highly specific binding & elution
TALON Magnetic Beads bind low to high-molecular weight his-tagged proteins with high specificity. Purified proteins are eluted in small volumes (50–200 µl), resulting in concentrated samples (up to 3 mg/ml). TALON Magnetic Beads are supplied as a 5% suspension with a demonstrated binding capacity of 750 µg of protein per ml of suspension.
Microscale purification with TALON Magnetic Beads can be used to screen expression levels or for protein-protein interaction studies. In addition, the use of TALON chemistry allows for seamless scale-up of purification of target proteins using our standard TALON resin.
Choice of native or denaturing purification conditions
TALON resin retains its protein-binding specificity and yield under a variety of purification conditions. It is stable under both denaturing and native (non-denaturing) conditions. Deciding whether to use native or denaturing purification conditions depends on protein location, solubility, accessibility of the histidine tag, downstream applications, and preservation of biological activity.
- Native conditions
Purifying a protein under native conditions is the most efficient way to preserve its biological activity, but requires that the protein is soluble. Advantages include:
- Eliminating the renaturation step at the end of the purification, saving time, and preventing significant loss of activity
- Retaining the ability to copurify enzyme subunits, cofactors, and associated proteins
- Denaturing conditions
Because proteins that are overexpressed in prokaryotic systems sometimes form insoluble aggregates called inclusion bodies, you may need to purify proteins under denaturing conditions—using strong denaturants such as 6 M guanidinium or 8 M urea to enhance protein solubility. Advantages include:
- Complete solubilization of inclusion bodies and his-tagged proteins
- Improved binding to the matrix and reduced nonspecific binding, due to full exposure of the histidine tag
Use of reducing agents
Purification with TALON resin may be carried out in the presence of β-mercaptoethanol, but not DTT (dithiothreitol) or DTE (dithioerythritol), to preserve reduced sulfhydryl (-SH) groups that are important for the biological activity and structure of a given protein. TALON provides higher yields than Ni-NTA in the presence of β-mercaptoethanol.
- Quick and easy separation of his-tagged proteins
- Highly-selective TALON chemistry for increased purity
- Eluted samples ideal for small-volume applications
TALON Magnetic Beads provide simple, effective separation of recombinant his-tagged proteins for a wide range of applications, including:
- Microscale purification of his-tagged proteins
- Studying protein structure and function
- Preparing proteins for X-ray crystallography
- Performing assays that detect protein-protein and protein-DNA interactions
- Immunization to raise antibodies against a protein of interest
- Screening for protein expression
- Optimizing purification conditions for scale-up with TALON Resin
TALON Magnetic Beads can also be used to immobilize 6xhis-tagged proteins for affinity chromatography, in order to:
- Analyze protein-protein and protein-nucleic acid interactions
- Purify untagged subunits or nucleic acids that interact with the immobilized protein
- Perform antibody purification
- Study interactions between ligands and receptors
- Choice of native or denaturing conditions to purify proteins
- Directed presentation of 6xhis-tagged proteins increases reproducibility and signal-to-noise ratios
- Varying the amount of beads per well allows for a wide range of binding capacities
- A powerful tool for analyzing interactions between biomolecules
The compatibility of common purification reagents with TALON resin are as follows:
|Beta-mercaptoethanola||10 mM (with caution)|
|CHAPSb||1% (with caution)|
|Guanidine hydrochloridea||6 mM|
|Imidazoled||200 mM at pH 7.0–8.0, for elution|
|SDSb||1% with caution|
a Use resin immediately after equilibrating with buffers containing these reagents. Otherwise, the resin will change color. Do not store resin in buffers containing these reagents.
b Ionic detergents like CHAPS (3-[(3-Cholamidopropyl)-dimethylammonio]-1-propane-sulfonate), SDS (sodium dodecyl sulfate), and sarkosyl are compatible up to 1%. However, due to their charged nature, you should anticipate interference with binding, even at low concentrations.
c Ethanol may precipitate proteins, causing low yields and column clogging.
d Imidazole cannot be used at concentrations higher than 5–10 mM for loading his-tagged proteins, because it competes with the histidine side chains (imidazole groups) for binding to the immobilized metal ions.
e Tris coordinates weakly with metal ions, causing a decrease in capacity.
Reagents incompatible with TALON resin
The following reagents are incompatible at any concentration:
- DTT (dithiothreitol) and DTE (dithioerythritol)
NOTE: Using strong reducing agents will interfere with cobalt metal ion binding to the resin.
- EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid), and polyethyleneimine (PEI)
NOTE: Although you can use EDTA at indicated points, it must be removed from the sample by gel filtration before applying the sample to TALON Resins.
Additional product information
Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.
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