TALON CellThru is a novel IMAC resin for purifying his-tagged proteins from crude cell lysates, sonicates, and fermentation harvests. This resin uses the same proprietary ligand as our TALON Metal Affinity Resin, but has larger beads (300–500 µm) which permit cellular debris to flow through without centrifugation. TALON CellThru Resin captures his-tagged protein directly from crude lysates in one quick step, minimizing protein degradation and generating higher yields of purified protein than conventional strategies.
TALON CellThru Resin is ideal for purifying membrane-bound proteins or multi-protein complexes, which typically pellet with the cell debris during the lysate clarification step and so are not available for binding to the resin. Because crude, nonclarified lysates can be applied directly to the resin, you can skip the centrifugation step, allowing membrane-bound proteins and multi-protein complexes to remain in the lysate where they can bind to the resin (see example).
The resin consists of large agarose beads that can be packed into standard chromatography columns with large-pore frits to prevent column blockage. Particulate material flows between the beads while the soluble protein product is captured by the ion-exchange functional groups attached to the beads. The resin is ideal for use in expanded bed chromatography for recovering even higher amounts of protein and is recommended for applications such as high-throughput purification of his-tagged proteins from crude extracts. We also offer CellThru 10 ml Disposable Columns (with a filter pore size of 90–130 µm) that allow cellular debris to flow through easily.
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.
- 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 (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.