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PI (isoelectric poin)

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Based on predicted pI (for example, a pI for turnip peroxidase is 9.10), how can you use DEAE-sepharose to further purify peroxidase?

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Please see the attached file.

The question you are asking is related to the separation of proteins using ion exchange chromatography. In this technique, one takes advantage of differences in the overall electrical charge on proteins due to their amino acid side chains, to preferentially bind some protein molecules more strongly than others to a resin inside an ion exchange column (a glass cylinder filled with resin packing amterial in a buffer). The protein of interest must have a charge opposite that of the functional group attached to the resin in order to bind. Because this interaction is ionic, binding must take place under low ionic conditions (ie. the solution containing the proteins and that in the column is of low ionic strength). Elution (removal of the proteins that are bound to the column material) is achieved by increasing the ionic strength to break up the ionic interaction, or by changing the pH of the protein (which changes or neutralizes the protein charges so they will let go of the column material).

In general, proteins carry both positive and negative charges in their amino acid side ...

Solution Summary

A discussion on how to purify peroxidase.

See Also This Related BrainMass Solution

pH and buffer

You have a protein that is well behaved at pH=7.0 (The ph of the buffer is 7.) and the pI of your protein is equal to 4.3 (pI=4.3). You have decided to use ion exchange chromatography to help isolate your protein.
1.Using this information, would you use a cationic column (anion exchange) or an anionic column (cation exchange)? Why?
2.What residues in your protein do you think would be in a majority?
3.Same question as in question 1 with the exception that your protein has a pI=9.2.

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