Purification of Synthesized Protein using Reverse His-tag Purification
The following protocol is designed to rapidly purify analytical amounts of translated protein from a PURExpress reaction (see Figure 3 on the main product page). It requires the target protein be less than 100 kDa in molecular weight and not capable of binding to IMAC resin. In practice, proteins less than 60 kDa are more readily purified using this procedure than proteins near the MW cut-off of the spin-column membrane. Additional equipment is necessary and includes: Ni-NTA Agarose (Qiagen), Amicon Ultracel 0.5 ml-100K spin concentrators. An example is illustrated in Figure 4 on the main product page.
- Add 10 mM magnesium acetate or other diluent to the reaction to increase the volume and make handling of the sample easier.
We recommend a minimum volume of 100 μl after dilution to minimize losses during purification. If the target will be too dilute after addition of the diluent, we suggest a larger reaction volume be used. Use of concentrated NaCl to dilute the reaction may help dissociate complexes between the target protein and translation factors. The NaCl will remain, however, after the final elution and downstream applications may require microdialysis. We suggest limiting the final concentration of NaCl to ≤ 0.4 M after dilution. Additionally, magnesium acetate should be included to keep [Mg2+] close to 10 mM.
- Apply the diluted reaction mixture to a Amicon Ultracel 0.5 ml-100K spin concentrator (0.5 ml maximum load volume) and centrifuge for 30–60 min at 15,000 x g at 4°C. If necessary, higher centrifuged forces can be used to push the mixture through the membrane up to the recommended limit of concentration used.
- Transfer the permeate/flow-thru to a new tube, preferably a 2-ml round-bottom microfuge tube with a leak-proof cap.
- Add 0.25 volumes Ni-NTA Agarose and mix thoroughly for 30-45 min at 4°C to allow His-tagged components to bind the resin.
- Apply the reaction mixture slurry to an empty Bio-Rad micro-spin column and centrifuge for 2 min at 1500 xg at 4°C.
- Collect eluate containing purified protein and proceed with experimental analysis.