[RTG]

I am attempting to answer a question that asks how to separate 3 different proteins with the following characteristics.

Protein 1 has a pI=5.3, MW=445kDa
Protein 2 has a pI=10.0, MW=12.kDa
Protein 3 has a pI=8.4, MW=450kDa

I am considering using an anion exchanger with DEAE-Cellulose resin and using a running solution with pH 7 and a Tris buffer. Protein 2 would carry a greater positive charge and would elute first followed by protein 3. Protein 1 would carry a negative charge and would interact with the stationary phase and elute last. I am unsure if my method is correct and I would appreciate anyone with experience in chromatography providing any assistance.

[jdy07]

You are correct about the relative charges of the proteins and that anion exchange would help you separate the proteins. So, I'll give you a couple of hints:

1) How does the anion exchange resin work? In other words, what property of the DEAE-cellulose allows for separating the proteins?  When you figure the mechanism of separation, determine if all of these proteins will interact with the anion-exchange column.

2) determine if this can all be done in one purification step.