I mean, after doing affinity chromatography with nickel beads, we do SDS-PAGE, then why do we need to proceed to ion-exchange chromatography? Then another SDS-PAGE, then we proceed to size exclusion chromatography, then another SDS-PAGE. Why so many steps? Any reason?
Thank you.
Imac is a pseudo affinity technique, which transfers to several proteins binding to the adsorbent. To separate these proteins it is common to run iex. Two steps are in general enough.
Thanks Omar for the answer. But after doing IMAC, we can use imidazole to obtain the protein, right? Therefore why is the need for ion-exchange chromatography after IMAC ?
It is only neccesary if the protein is not pure enough for your needs.
If you think one step is enough, explain why. How do you plan to determine purity? What (mess of stuff) are you purifying from? ..not passionate about all the 'steps'? Invent a new one.
Irene calm down, I'm just asking a question. Don't answer if you're not answering the question. Thank you.
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