Insoluble protein after ammonium sulfate precipitation - (Sep/03/2015 )

I obtained my crude by homogenizing my sample with a pH 6 buffer solution. Then add 80% ammonium sulfate saturation (separate into 3 parts and added slowly) to the crude solution, stirred for 6 hours on ice. After centrifuge, the pellet was resuspended with the same buffer and was subjected to dialysis against pH 6 buffer solution (with a minimum of 3 changes of the buffer, overnight). 

However during dialysis, clumps start to form and you could see that there is a two layer separation (the clumps which are cloudy and clear solution). Is this a form of protein insolubility and is there anyway I can overcome this? I have tried using a different pH buffer (pH 4.5) for dialysis but it gave the same outcome.

there will almost always be proteins which denature permanently when precipitated, especially when chemically induced (i.e. ammonium sulfate, acetone, ethanol, etc).

sometimes the denatured protein will also "carry down" some native proteins when centrifuged. you can avoid this and ensure that only denatured proteins pellet by homogenizing the solution with a loose fitting dounce homogenizer.

another possible cause of the clumps is isoelectric precipitation. most proteins have a pI between 4 and 6. the closer the pH is to the pI, the less soluble the protein. since you are working within the range, you may be seeing this.