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Exercise 3 of 6
Ion exchange chromatography of a simple mixture of three proteins

Now you are going to try to purify the mixture using ion exchange chromatography. The medium that you will use is DEAE-cellulose. DEAE- stands for diethylaminoethyl-, a tertiary amino group which will carry a positive charge except at very alkaline pH values. So your medium will be positively charged and therefore will bind proteins which carry a net negative charge. You need to think carefully about the isoelectric point (pI) of a protein and the pH value of the buffer in which the protein is suspended. If the pH of the buffer is the same as the pI of the protein, then the protein will carry zero net charge. (Not zero charge - the positive charges exactly equal the negative charges and so there is a net zero charge.) If the protein is suspended in a buffer whose pH value is less than the protein's pI, the protein will pick up protons from the buffer and hence have a net positive charge. Conversely, if the protein is suspended in a buffer more alkaline than the protein's pI, the protein will lose protons to the buffer and become net negatively charged. This is an important concept in understanding how proteins interact with ion-exchange media. Here is the 2-dimensional electrophoresis gel of the mixture. Look at it again.

2D gel

If this mixture was applied to a column of DEAE-cellulose at pH 7.0, which proteins would bind to the column?

Which proteins would bind at pH 8.0?

Which proteins would bind at pH 6.0?

Abandon Step menu
Now test your predictions. If you have not already done so, click on the Quit menu and select Abandon this step and continue.
Ion Exchange menu
 

Then click on the Separation menu and select Ion exchange chromatography...

Ion Exchange Dialog

Click on the DEAE-cellulose and salt gradient radio buttons. Then click on the OK button.

pH Dialog
 

Click in the edit box, type 7.0 (or use the direction buttons) and then click on the OK button.

Gradient Dialog In order to elute any proteins bound to the column, you need to wash the column with a buffered salt solution. If the concentration of this solution increases with time, then the proteins will be eluted in order of their net charge.

 So you need to tell the program about this solution. Set the start of the gradient to 0.0 and the end of the gradient to 0.5 molar (if they are not already set to these values). Now click on the OK button.


Once again, the computer will simulate the behaviour of the mixture under the conditions that you requested. Look at the elution profile. As before, the computer has measured the absorbance of each fraction at 280nm. It has also measured the salt concentration in each fraction. The salt gradient starts to emerge from the column at fraction 32, so anything present in the 'earlier' fractions represents material that did not bind to the column, but was washed straight through.

How many peaks are there? Examine the material in each by 2-dimensional electrophoresis. Which protein is in which peak? Is this what you would have expected?

 Use the Quit menu to Abandon this step and continue. Then try repeating the experiment at pH 8.0 and 6.0. (You may need to adjust the salt gradient to ensure that all three proteins are eluted.) How do the results differ? Can you explain the differences?


Do you think that you could purify protein 2 in a single step by gel filtration or ion exchange chromatography?

Can you think of a method for purifying protein 2 from this mixture using the methods that you have tried so far?

Would it make a difference if you changed the order in which each method was used?

Try to purify protein 2.