Hello Ahmed,

First two questions:

- Why do you need to concentrate and buffer exchage your material before loading it onto the Ni-NTA column ? Does the medium contain components interfering with binding of the presumably His-tagged protein to the Ni-NTA resin ? If not, you could just load the cleared supernatant directly on the Ni-NTA resin and elute the protein in a small volume.

- How come precipitation and dialysis should take one week ? Ammonium sulphate precipitation will take at most a couple of hours, leaving sufficient time for the precipitate to form plus 30 min centrifugation. Dialysis with two >4hr changes of buffer will take at most one day including one overnight step. In addition, it would probably be unnecessary, since binding to Ni-NTA is not affected by ionic strength.

If you want to concentrate large volumes by ultrafiltration, the way to do so is probably to use tangential ultrafiltration cassettes, which prevent clogging and membrane polarization. These are however significantly more costly than ultrafiltration devices based on centrifugation.

Yes I agree, the simplest path is ammonium sulfate, re-suspend pellet, and load the column. If not this, only tangential flow makes sense at this scale.

With the complete agreement to what Dr.Pierre Béguin said,

I would use batch purification format for affinity (IMAC) capturing of POI if a large amount of protein sup is the case. However, amicon stirred cell ultrafiltration device with the upper limit of 400mL volume may help for buffer exchange application but it is not needed for improved binding affinity at IMAC resins...