Hi, I am trying to exchange the buffer and then concentrate a protein solution which it has just one pure peptide. The volume after dialysis is about 2 ml and i tried to percipitate the protein using ammonium sulphate. I added slowly1.43g (NH4)2SO4 to the solution in the fridge while stirring to get 100% saturation but after one hour I just can see some extra crystals of salf not my protein! Any idea would be appreciated.
So, you have exchanged the buffer and now you want to concentrate the protein, is that right?
I have a couple of thoughts: For one, after ammonium sulfate precipitation, you will probably have to exchange the buffer again, depending on what you want to do with the protein. So, you probably could have done the precipitation before the buffer exchange. Second, the concentration of ammonium sulfate at which a protein precipitates is specific to the protein. Plus, it is not always that efficient. So, if you want to use ammonium sulfate, see if you can find the precise concentration required for your protein. If you have already done this, it could be that the starting concentration of the protein is too low or you didn't give it enough time to precipitate.
My preferred method to do a buffer exchange and concentration at the same time is to use a centrifugal concentrator such as an Amicon Ultra (link
). You load the device with your protein solution, spin until the volume is reduced to about one-tenth the original, add the new buffer up to the max volume of the device, reduce the volume, and repeat with another volume of buffer. When complete, you will have your protein in a small volume but in the buffer of your choice. The Amicon devices have very high protein recovery, by the way.