His-Tagged Protein Purification from Bacterial Cultures—TALON Single Step
TALON Single Step Columns simplify purification of his-tagged proteins. These columns combine our exclusive TALON xTractor Buffer with the patented TALON resin, to consolidate the preliminary purification steps: cell lysis, centrifugation, and resin binding. These columns provide a convenient method of purifying material for preliminary characterization of your protein of interest.
Easy, Fast, and Pure
The entire process, from culture to purified protein, can be completed in less than 1 hour.
Add the culture containing your his-tagged protein to the column and then mix for 20 minutes. The column contains a dried version of TALON xTractor Buffer, which lyses cells and releases your his-tagged target protein. Our proprietary TALON Resin binds his-tags with higher specificity than any other IMAC resin, so you are assured of obtaining a high degree of purification.
After the unbound cellular debris is washed from the column, your highly pure his-tagged fusion protein is eluted using standard conditions.
Looking to purify smaller amounts of protein? The 5-ml columns routinely provide up to 0.5 mg of purified protein and the 20-ml columns regularly provide up to 3.0 mg of purified protein.
Value and Versatility
These time-saving columns are more convenient than the typical method of extracting and purifying his-tagged protein over a standard TALON Resin Column. Any 6xHis-, 6xHN-, or HAT-tagged protein can be purified using either a gravity flow or a spin column method. It is easy to run several columns in parallel at room temperature, to isolate various proteins at the same time.
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.