Results and analysis:

1. 1. Switch ON the UV/Visible spectrophotometer and allow it 30 min warm up.

2. Set the instrument to absorbance mode and wavelength to 280 nm.

3. Zero the reading taking wash buffer as a blank.

4. Measure the absorbance of all the fractions (do not wash the cuvette in-between the readings).

5. Record the absorbance in an observation table (Table 25.2).

Table 25.2: Table for recording absorbance of collected fractions

1. 6.  Plot the absorbance values against the fraction number to obtain the chromatogram.

7.  The purified His-tag fusion protein usually elutes in fraction no. 2 – 5.

Notes:

1. 1.  Batch purification:

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

   b.  Pipette out the required volume of the slurry (5 ml packed volume) in a polypropylene centrifuge tube.

   c.  Centrifuge at 750 × g for 2 minutes and gently aspirate the supernatant.

   d.  Add three bed-volumes of the binding buffer, resuspend the resin by inverting the tube and gently tapping it, centrifuge at 750 × g for 2 minutes and aspirate the supernatant.

   e.  Repeat step ‘d' two more times.

   f.  Add the clear lysate (obtained in step 14 of ‘Procedure' section) to the Ni-NTA resin and mix gently by shaking the tube on a rotary shaker at 4 °C for 60 minutes.

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

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

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

   j.  Wash the column 4 times with 2 bed volumes of wash buffer.

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

   l.  Perform analysis as discussed in ‘Results and Analysis' section.

2.  Some amount of the protein of interest may be present in insoluble form in the pellet. For recovery of this fraction, the protein can be solubilized using denaturing conditions.

3.  Before starting purification, it is recommend to run an SDS-PAGE with the lysate to ensure that the protein was expressed by the bacteria (or whatever expression system was used) in good amount and is present in the lysate.

4.  The NTA resin appears light blue-green when charged with nickel ions and turns white when stripped of the ions.