Hello to all! I've just recently sign up as a user here ^0^ please do tell me if I got anything wrong anywhere.
So, onto the main topic of discussion is that I am currently doing protein purification for his tag purification and anion exchange column for the next step of purification. I have no problem when using the nickel affinity column, but when it comes to the ion exchange column, it seems that most of the protein of interest get out in the flow through fraction rather than the elution fraction. I don't know why my protein does not bind to the ion column even though the buffer's pH and salt concentration would not disturb the protein binding. By the way, the details are as follow:
Protein pI: 6.3
Protein buffer salt (NaCl) concentration: 10mM pH 9
Ion exchange column buffer A salt concentration: 20mM pH9
Ion exchange column buffer B salt concentration: 2M pH9
I've done dialysis after the nickel affinity column, changing the salt concentration from 150mM to 10mM
Thank you in advance!!