The salts should be removed from the solution contains proteins and salts (calcium oxide/ sodium chloride) by diafiltration process. My question is
1) Can we use the ultrafiltration membrane for diafltration process or is there any separate membrane specified for diafiltration?
2) What will be the molecular cut off of membrane for separation of salts and proteins?
3) How to confirm that the salts are completely removed from the solution?
Using ultrafiltration, you first concentrate the protein as much as you reasonably can, then redilute it to the original volume with a buffer lacking the salts you want to remove, then concentrate it again. Repeat the process if needed.
The salts pass freely through the membrane but the protein does not. So, as an example, suppose you reduce the volume of the protein solution in the first step by 10-fold, then redilute to the original volume. The concentration of salts will be reduced by 10-fold. Now repeat the process of 10-fold concentration and redilution and the salts will be reduced 100-fold.
Another procedure to get rid of the salts is dialysis.
A fast way to do diafiltration is with a specialized diafiltration unit. This is best applied to large-volume samples, whereas the ultrafiltration and dialysis methods are better for small-volume samples.
I'm agree with the answers of Omar.
Just one more suggestion:
If your sample volume is not so high, you can also perform a desalting processes.
Dialisis and ultrafiltration dilute your salt concentration in relation to the dilution factor.
E.g;
- dializing 1 step 10ml vs 1liter --> 100times dilution (so if you start from 300mM of salt) in the final sample you still have 3mM of Salt.
- ultrafiltration 1 step of concentration/dilution from 20ml to 1 dilute the salt only 20 times and for strong salt removal you need to perform more cicle of concentration/dilution. Play attention to not concentrate to much the sample because in some case (depends from protein stability and properties) it can drive to protein aggregation.
Desalting is fast and if you do not exceed with volume and sample amount generally in one step allow to complete remove salt.
Up to 2.5ml of sample you can use PD-10 desalting coloumn and gravity flow.
Up to 15ml you can use desalting 26/10 coloumn (Ge) connected with a chromatograph for protein purification (eg. AKTA). In this case looking A280nm and conductivity you will easly see the protein and salt profile and check if the salt removal it is succeded.
good luck
Manuele
Good answers!!
I fully agree with Manuele. Just a comment, Dialysis and ultrafiltration take long time compared to Desalting by gel filtration and they are less efficient than this one. Gel filtration is not suitable if the removing of the salts must be slowly. For example, if you want to remove the urea used to solubilizing inclusion bodies, desalting on gel filtration maybe lead to agregation or desnaturalization of the protein. In this sense, to use dialysis is better.
Best regards, Oscar
Hi everyone,
Now i get clear idea. Thank you for your answers.
One more question related to this...
Which technique (Dialysis, Ultrafiltration or gel filtration chromatography) is viable and economic at industrial scale level?
I believe that diafiltration by tangential flow filtration is the method best-suited to buffer exchange at industrial scale.
Dialysis : Difficult to do scale up GFC : Large resin volume required since amount of the solution process is less compared to TFF and more unit operation time, ultimately increase the product cost. While TFF easy to do linear scale up and we can reduce the process volume so it is easy to handle also. But if your protein is too sensitive to shear one can go with GFC by compromising the cost and time part.
TFF (tangential flow filtration) is it certanilly the easiest to scale up is your protein is stable under concentrarion/dilution cicle.
However you can certanilly as first choihe try asUltrafiltration work in small scale (that in principle is similar to diafiltration) and if it work well, try to use TFF for the scale-up.
good luck
Manuele
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