483 Lecture 5 - Glutamate Oxaloacetic Acid Transaminase (GOT) Purification -- Expt 3 Part B
2. Dialysis of Proteins
After a protein has been ammonium sulfate precipitate and taken back up in buffer at a much greater protein concentration than before precipitation, the solution will contain a lot of residual ammonium sulfate which was bound to the protein. One way to remove this excess salt is to dialyze the protein against a buffer low in salt concentration.
This graphic illustrates the dialysis process. First, the concentrated protein solution is placed in dialysis bag with small holes which allow water and salt to pass out of the bag while protein is retained. Next the dialysis bag is placed in a large volume of buffer and stirred for many hours (16 to 24 hours), which allows the solution inside the bag to equilibrate with the solution outside the bag with respect to salt concentration. When this process of equilibration is repeated several times (replacing the external solution with low salt solution each time), the protein solution in the bag will reach a low salt concentration:
The graphic illustrates the complete dialysis process, except for it suggests you do this with distilled water. Really you want to do this process with buffer to prevent the protein from denaturing due to the fact that distilled or deionized water is too low in salt and may have an undesirable pH for your protein, which may cause it to denature.
In fact, dialysis is a good way to exchange the buffer the protein is in at the same time you get rid of excess salt. For example, in the expt we are doing this week in lab, the GOT after ammonium sulfate precipitation contains a mixture of buffers as well as excess salt. So we use the buffer we want for the next step in the purification, which is ion-exchange chromatography, as the external solution during dialysis. After the 3 step dialysis process where the protein solution is dialyzed against the starting buffer for the ion-exchange chromatography step, not only will the salt be removed but the protein will now be in the buffer needed for the next step and ready to go.
Sometimes, proteins will precipitate during the dialysis process and you will need to centrifuge the solution after dialysis to remove any particles which would interfere with the next step - such as ion-exchange chromatography where particles would clog the column and prevent the chromatography step from working.
In addition, you may lose enzyme activity during dialysis. So it is a good idea to keep some of your protein solution as a sample before it is put in the dialysis bag so that it can be assayed for enzyme activity before and after dialysis.
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