Procedure:

Packing of the column (all the steps are to be carried out at 4 ºC )

1. 1.  Prepare all the reagents and solutions and keep them at 4 ºC .

2.  Take out the bottle of the Ni-NTA resin and resuspend the resin by inverting the bottle and gently tapping it.

3.  Mount the polypropylene column on the laboratory stand in the cold room.

4.  Gently pour the Ni-NTA resin to obtain a packed bed volume of ~5 ml .

5.  Allow the storage buffer to drain down the column.

6.  Equilibrate the column with 3 bed volumes of the binding buffer, keep sufficient binding buffer above the resin bed and stop the flow.

Preparation of cell lysate (all the steps are to be carried out at 4 ºC )

1. 7.  Collect the bacterial culture (after His-tag fusion protein expression) in a centrifuge tube.

8.  Harvest the cells by centrifuging at 4,500 × g for 10 minutes at 4 ºC.

9.  Resuspend the cell pellet in 10 ml of binding buffer.

10.  Add 10 mg of hen egg white lysozyme (final concentration: 1 mg/ml ) and incubate in ice for 30 min .

11.  Sonicate the lysozyme treated bacterial suspension with six 10-seconds bursts of 200-300 W each with a 10 second cooling after each burst. If the bacterial lysate appears to be viscous, add Rnase A (10 μg/ml ) and Dnase I ( 5 μg/ml ) and incubate on ice for 15 minutes.

12.  Centrifuge the lysate at 10,000 × g for 20 minutes to pellet down the cellular debris.

13.  Transfer the supernatant to a fresh tube (Notes 2 and 3).

14.  Centrifuge the supernatant collected in step 13 at 25,000 × g for 5 minutes to remove the insoluble aggregates and obtain the clear lysate.

Protein purification (all the steps are to be carried out at 4 ºC )

1. 15.  Start the flow of the column and load the entire clear lysate to the column.

16.  Allow the cell lysate to enter the column completely.

17.  Wash the column 4 times with 2 bed volumes of wash buffer (i.e. a total of 8 bed volumes).

18.  Add 0.5 bed volumes of the elution buffer 10 times collecting the 0.5 ml fractions each time.

NTA resin regeneration

1. 19.  Wash the resin with the following solutions without allowing the resin to dry:

   a.  2 bed volumes of stripping solution

   b.  2 bed volumes of water

   c.  3 bed volumes of 2% SDS

   d.  1 bed volume each of 25% ethanol, 50% ethanol, and 75% ethanol

   e.  5 bed volumes of ethanol

   f.  1 bed volume each of 75% ethanol, 50% ethanol, and 25% ethanol

   g.  1 bed volume of water

   h.  5 bed volumes of 0.1 M EDTA, pH 8.0

20.  After step 1, the NTA resin is completely stripped of the nickel ions (Note 4).

a.  For long-term storage, wash the uncharged resin with 5 bed volumes of 20% ethanol and store at 4 °C.

21.  For immediate use, recharge the column by adding 5 bed volumes of 100 mM NiSO₄·6H₂O. and washing with 10 bed volumes of binding buffer. The column is charged again.