You are trying to purify a protein that is soluble in a solution of 2 M ammonium sulfate. After centrifugation to remove other proteins that have precipitated at this high salt concentration, you recover the supernatant to assay the target protein’s activity in a cell culture system.

(a) Explain why the cells die when incubated with the supernatant.

(b) What procedure could you perform to correct the problem? (Hint: See Section 2-1D).

If the dissolved salt concentration outside of the cell is higher (hypertonic environment), water will diffuse "out" of the cell, causing the cell to dry and shrink, and cellular metabolism to stop. Thus, high salt concentrations would completely inhibit or kill cells.

The supernatant contains the desired protein as well as high concentration of ammonium sulfate salt. This high concentration of salt solution when added to the cell culture, ceases the cell’s metabolism which will ultimately kill all the cells in the cell culture.

The laboratory technique of dialysis is based on the diffusion of solutes. Solutes smaller than the dialysis membrane's pore size easily exchange between the sample and the bulk solution until equilibrium is established.Larger substances cannot pass through the membrane and stay where they are.

Dialysis is particularly useful for separating large molecules from smaller molecules, such as proteins or nucleic acids.

The supernatant contains high concentration of ammonium sulfate. This is first removed by using dialysis process. The supernatant is poured in the dialysis membrane and the membrane is then placed in a beaker containing water. The salt will move out but the large protein molecules will stay inside the dialysis membrane. This will reduce the salt concentration in the supernatant and can now be added to the cell culture.