Protocol for purifying His-tagged proteins using Proteus IMAC kits

Under NATIVE conditions

These protocols refer to the purification of active folded proteins. As strength of binding will be determined by length and accessibility of the His-tag the metal ion, buffer composition and pH and elution conditions, some adjustments of the procedure in this handbook is often required.

Ideally, the cell lysate should be made up in phosphate buffer pH 7.4-8.0 in the presence of NaCl (to reduce non-specific adsorption effects).

The following metal chelate buffers are proposed for your IMAC separation. The procedure to prepare these working buffer solutions from stock buffers supplied in the kit is shown in Table 1 (page 23).

Suggested buffers for NATIVE IMAC purifications:

All buffers contain sodium azide as a preservative.

  • Binding buffer: 50 mM sodium phosphate buffer pH 7.4, 300 mM NaCl, 10 mM imidazole.
  • Wash buffer: 50 mM sodium phosphate buffer pH 7.4, 300 mM NaCl, 30 mM imidazole.
  • Elution buffer: 50 mM sodium phosphate buffer pH 7.4, 300 mM NaCl, 300 mM imidazole.

Table 1 showing how to prepare your metal chelate binding, wash and elution buffers.

The two buffers provided in the kit are supplied as concentrated stocks. Always measure the pH of the working buffer solutions when they are prepared and adjust to pH 7.4 whenever necessary.

It is not uncommon for buffers to precipitate or freeze partially in the cold during long term storage or when laboratory temperatures drop at night. The buffers should be warmed up and they can be used once all the precipitate has re-dissolved.

Under DENATURING conditions:

Recombinant proteins often form insoluble inclusion bodies when they are expressed at high levels. These proteins can be solubilized easily in the presence of denaturants such as 6-8 M urea or 6 M guanidine hydrochloride. Additionally, a researcher may be choose to purify their recombinant protein under denaturing conditions if they wish to use the purified denatured protein for raising antibodies. Two buffer configurations can be used under denaturing conditions. One buffer system employs imidazole to competitively elute the target protein under denaturing conditions and the other buffer system uses a more acidic pH to elute the target protein in the absence of imidazole. Choosing either buffer system on page 25 will depend critically upon the nature of your target protein e.g. stability in acid environment.

Suggested buffers for DENATURING purifications:

(1) Imidazole Elution:

  • Binding buffer: 50 mM sodium phosphate buffer pH 7.4, 300 mM NaCl, 10 mM imidazole, 6-8 M Urea.
  • Wash buffer: 50 mM sodium phosphate buffer pH 7.4, 300 mM NaCl, 30 mM imidazole, 6-8 M Urea.
  • Elution buffer: 50 mM sodium phosphate buffer pH 7.4, 300 mM NaCl, 300 mM imidazole, 6-8 M Urea.

(2) Acid Elution:

  • Binding buffer: 50 mM sodium phosphate buffer pH 7.4, 300 mM NaCl, 6-8 M Urea.
  • Wash buffer: 50 mM sodium phosphate buffer pH 6.0, 300 mM NaCl, 6-8 M Urea.
  • Elution buffer: 50 mM sodium phosphate buffer pH 4.0, 300 mM NaCl, 6-8 M Urea.

N.B. Addition of urea will cause the pH to drop. Titrate the buffer with NaOH to bring the pH back to pH 7.4. The pH of those buffers containing urea should be checked and adjusted, if necessary, immediately before use.