483 Lecture 6 - Glutamate Oxaloacetic Acid Transaminase (GOT) Purification -- Expt 3 Part C
2. Ion Exchange Chromatography - Part B - Using Ion Exchange for Protein Purification
The process of using the ion exchanger, for example a cation exchanger, in a protein or enzyme purification goes like this:
- Equilibrate the cation exchanger with low salt concentration buffer of desired pH
- Apply the enzyme in same buffer at same pH and salt concentration
- Wash the unbound ions away using the equilibration buffer
- Elute the bound enzyme by increasing the salt concentration in the buffer
Here are illustrations showing these steps:
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- For enzyme binding, the protein solution is applied to Ion Exchanger at fixed pH and low [salt]
- For elution of enzyme from Ion Exchanger, a high [salt] is applied to column.
Generally, the ion exchanger is poured into a column before the protein or enzyme is applied and then after the binding and washing steps, the enzyme or protein is eluted into a fraction collector with the effluent or solution eluting from the column being monitored by an absorbance device for measuring the protein concentration in the effluent.
We will use a similar set up in the lab this week as described in other sections of this lecture.
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