483 Lecture 5 - Glutamate Oxaloacetic Acid Transaminase (GOT) Purification -- Expt 3 Part B
3. Alternative Methods for Desalting and Concentration of Proteins
There are several ways to get rid of excess salt in a protein solution. One rapid method is to use a small gel filtration column which contains a gel with very small pores which will only allow water and salt inside the gel particles and will exclude the protein. This method works very well and can be done at 4°C so that little or no enzyme activity is lost during processing. A small amount of dilution of the protein solution will take place during processing, but it is possible by this method to exchange the buffer and prepare the protein solution for the next step in the procedure. We will discuss the process of gel filtration in more detail in Week #8 in relation to estimating the native molecular weight of GOT.
Another way to both concentrate a protein and exchange the buffer, which completely avoids precipitation, is called ultrafiltration:
Ultrafiltration is done a device which can withstand high pressure. First, the ultrafiltration device is fitted with an ultrafilter membrane of the desired molecular weight cut off such that you protein of interest will be retain in the cell. Next, the pressure cell is filled with the protein solution and nitrogen gas at about 50 psi is applied while the cell is stirred gently at 4°C. After about 1 hour, the solution will be decreased in volume usually without loss of activity. To exchange the buffer the cell is filled with the desired buffer and the concentration process is repeated.
Back To: Lecture 5 - Week #5