Lab Three: ION EXCHANGE CHROMATOGRAPHY Part B

Part B: Ion Exchange Chromatography

So in Part A, we isolated the proteins, including rubisco, using Solubility.

So in Part B, what are we doing? Isn't that enough?

No...we want RUBISCO not the other mess!

What is important in this lab?

• What property is being used to separate the proteins?
• What charge do the beads carry?
• What charge is Rubisco?
• What is the purpose of the low-salt buffer? medium-salt buffer? high-salt buffer?
• Why do we collect all the flow through?
• Which flow through contains Rubisco?
• Why do we take the OD of all the samples?

Big Players at the Table:

Low Salt Buffer, Medium Salt Buffer, High Salt Buffer, and Proteins.

So through your research, you should have determined Rubisco has an overall NEGATIVE charge!

So we want to use a POSTIVELY CHARGED COLUMN! So what are we using to separate the proteins?? CHARGE!

Let's talk about the Ion Exchange Column...

The resin contains positively charged cellulose beads that hold onto the negatively charged proteins.

At this point, it's a battle of the strengths. Salt Concentration strengths, that is.

So we put in the supernatant and pellet (into different columns of course). Then we use the salt buffers to battle with the proteins.

Low Salt:

Competes with the very low charged proteins that have bound to the column. Those that have a very low negative charge will come out in the flow through of the low salt buffer.

Medium Salt:

Competes with the medium charged proteins on the columns. This knocks the medium negatively charged proteins into the flow through.

High Salt:

Competes with the high charged proteins on the columns. This knocks the strong negatively charged proteins into the flow through.

So where do we expect the Rubisco?

• Pellet or Supernatant?
• Low Salt?
• Medium Salt?
• High Salt?
• What does the OD reading tell you about the amount of protein in the flow through? Is it rubisco? Can we tell?

There's another part...keep on truckin'!