You can take a small sample of your preparation after dialysis and apply it to a desalting column (Gelfiltration, most suppliers offer them as small (1,5-ml-cup-sized) disposable prepacked columns usable in a centrifuge to fasten up the process). Now measure A280 of the desalted protein solution and of the dialysed protein solution. If the dialysate gives a appreciably higher A280 than the desalted protein solution, the difference in A280 is caused by residual imidazole. However, small differences might also be due to protein-binding to the desalting column.

Yes, you can estimate it. You need to calculate it depending on the volume of buffer you are using for dialysis.

I am just showing you one example:

If your elution buffer cotain 200mM immidazol, if you use 10mL protein(after purification) to dialyse with 2L of buffer with same condition expet immidazole, the immidazole of your protein buffer can be calculated by the following equation:

S1 x V1 = S2 x V2

where, S1 = intial concentration of immidazole, i.e., 200mM, V1 = initial volume of your sample, i.e., 10mL, V1 = final volume of your total sample, i.e., 2000mL+10mL=2010mL, and S2 is your final concentration of immidazole

, so you can calculate the concentration of immidazole after first dialysis. If you go for several dialysis, you can calculate by assuming the immidazole concentration for S1 which you get after 1st dialysis to calculate the concentration for 2nd dialysis.

Thanks Peter.

Thanks actually quite good way to estimate the imidazole concentration. will definitely give it a try.

Thanks.

Hi Sharoar

Thanks for your suggestion, but that how it works theoritically, but after dialysis i need to confirm that diaylsis went fine, and want to confirm it experimentally that ok, imidazole is gone from my sample.

Theoritically you are absolutely right..

Thanks again.