His-Tagged High-Throughput Micro-Scale Purification—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.
Highly Specific Binding & Elution
TALON Magnetic Beads bind his-tagged proteins ranging from low to high molecular weight 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 (nondenaturing) conditions (see flow chart). Deciding whether to use native or denaturing purification conditions depends on protein location, solubility, accessibility of the his tag, downstream applications, and preservation of biological activity.
Purifying a protein under native conditions (see example) is the most efficient way to preserve its biological activity, but requires that the protein be 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
Because proteins that are overexpressed in prokaryotic systems sometimes form insoluble aggregates called inclusion bodies, you may need to purify proteins under denaturing conditions (see example)—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 his tag
His-tagged proteins purified under denaturing conditions can be used directly in subsequent applications, or may need to be renatured and refolded. Protein renaturation and refolding can be performed prior to elution from the column. However, yields of recombinant proteins will be lower than under native conditions, because urea and guanidinium molecules compete with histidines for binding to metal.
Use of Reducing Agents
Purification with TALON Resin may be carried out in the presence of β-mercaptoethanol (see example), but not DTT or DTE, 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 (see example) in the presence of β-mercaptoethanol.