Thoroughly dialysis should completely replace your buffer (i.e., remove imidazole). Imidazole absorbs at 205-210 nm, so absorption would be able to give you an idea of how much imidazole you still have in your sample.

Since imidazole passes readily through dialysis membrane, if you allow sufficient time for the dialysis to reach completion (overnight) and provide sufficiently vigorous mixing by magnetic stirring, the concentration of imidazole remaining in the sample can be calculated as the initial concentration multiplied by the ratio of the sample volume to the dialysis bath volume. Replacing the dialysis bath with fresh solution and repeating the dialysis can reduce the imidazole to a negligible concentration.

You can remove most of the imidazole more quickly using 2 serial desalting spin columns, such as PD-10 columns (Cytiva), although you will lose some of the protein this way.

You can use gel filtration chromatography on a preparative scale column to remove the imidazole. The protein will elute ahead of the salts.

Another way to remove most of the imidazole is to concentrate the protein with a centrifugal ultrafiltration device, then dilute it back to the original volume with imidazole-free buffer. Repeat the process again for more thorough removal. If each time the protein volume is reduced 10-fold and then rediluted, two cycles will remove 99% of the imidazole.

Dear Hong Thai Le Nguyen

if the dialisys is peformed with right cut-off and enough time to reach the equilibrium theoretically as suggested from Adam B Shapiro he final concentration of imidazole depends could be theorerically calculated from the relationship from the volume and buffer sample.

To remove completelly the imidazole more steps with a lower volume can be better than a single step with a hugh volume.

Eg

1 Step 1ml agaist 1 liter

starting concentration 300mM imidazole

starting sample volume 1ml

buffer volume: 1liter (1000ml)

Final imidazole concentration --> 300x1/(1000+1)= about 0,3mM

2 step 1ml agaist 100ml

starting concentration 300ml

starting sample volume 1ml

buffer: 100ml x 2 steps

1°step --> final concentration 300x1/(100+1)= about 3mM

2°step --> final concentration 3x1(100+1)= 0,03mM

so with less buffer (200ml instead 1liter) you will reduce the buffer concentration 10 time more. Of course this apporach need more time.

An other alternativec that i like is desalting (using PD-10 gravity flow for samples up 2.5ml or HI-PREP 26/10 couple with AKTA for samples up to 12ml, which if well performed remove completelly the Imidazole.

best regards

Manuele