Well it depends on the sample you are processing. The procedure you mention is really mild. After processing sample, to obtain reproducible results, and somewhat wash the column I prefer to remove th Ni ions after each run and charge the column with new ions for new batches.

Dear Abir

on my blog ProteoCool ( https://proteocool.blogspot.com/p/blog-page.html) you can find a video

ProteoCool n°25 (A simple way to clean and regenerate  the IMAC coloumns)

where you can find some indication about buffers and volumes that i'm using to regenerate IMAC coloumns

ciao

Manueele

I would only ever use this technique for regeneration if I used the resin for purifying exactly the same protein.

Otherwise I would follow what Omar Gonzalez-Ortega suggested which is fully regenerate and recharge.

1.

I normally "fine" the resin first, a couple of repeated suspensions in 25mM HEPES pH 7.5, 125mM NaCl.

To do this allow the resin to just settle, then pour off the supernatant, then repeat. Don't allow to settle for longer, you're looking to remove small insolubles and resin fragments. Your resin will have much improved flow characteristics, the small insolubles don't fill any interstitial spaces (void volume) impairing the flow. This becomes more important with more usage of the resin.

2.

Then strip with 5 column/resin volumes of 25 mM HEPES pH7.5, 125mM NaCl, 100 mM EDTA,

3.

Remove non-specifically bound material by applying 5 column volumes of 0.2M NaOH and leaving overnight at RT.

4.

Wash with at least 10 column/resin volumes (each volume applied individually) of 25 mM HEPES pH7.5, 125mM NaCl. I normally put a bolus of 1 CV of 250 mM HEPES pH 7.5 first.

5.

Recharge with 25 mM HEPES pH7.5, 125mM NaCl and 100mM NiSO4 or NiCl2.

If you look to get the same intensity colour charge of nickel on the column the use of buffering is essential, I don't find the same if you use only the nickel salt to recharge (probably down to their slight acidity in solution).