Hello! I am purifying a 4.4kda protein. This protein is the C-terminus of a larger 25kda protein. So, in order to isolate it, we inserted a thrombin cutting site in between the amino acids to be cut. After cutting, we expect to get a ~20kda with His tag and my protein 4.4kda. We tried to pass it through Ni-beads so that the 20kda with the His tag remains in the beads and the 4.4kda will be in the flow through. However, we tried many ways but my protein seem to disappear. We also tried using ultrafiltration but we can't see my protein. Does someone have an experience on this? any advice?