Protein purification always begin with intact tissue  Disrupt  Blender, homogenizer  Remove debris  Centrifugation  Precipitate/concentrate  Ammonium sulfate  Remove salt  dialysis  Purify  Chromatography  Analyze  Activity, molecular weight

Salting in / Salting out  Salting IN  At low concentrations, added salt usually increases the solubility of charged macromolecules because the salt screens out charge-charge interactions.  So low [salt] prevents aggregation and therefore precipitation or “crashing.”  Salting OUT  At high concentrations added salt lowers the solubility of macromolecules because it competes for the solvent (H 2 O) needed to solvate the macromolecules.  So high [salt] removes the solvation sphere from the protein molecules and they come out of solution.

1. salting in - proteins are less soluble at very low salt concentrations (low ionic strength); salt ions helps shield protein's multiple charged groups 2. salting out - proteins are also less soluble at salt concentrations (high ionic strength) because the salt ions bind most of the water molecules 3. These two effects are different for different proteins ==> fractionate by raising the [salt] just below point where protein to be purified becomes insoluble, removed other precipitated proteins by centrifugation, then raise the [salt] to precipitate protein and collect it by centrifugation.

A protein in a buffer solution is very highly hydrated, in other words, the ionic groups on the surface of the protein attract and bind many water molecules very tightly: This graphic illustrates how proteins in solution are hydrated by water molecules.

When a lot of salt, such as ammonium sulfate, is added to the protein solution, the salt ions attract the water molecules away from the protein. This is partly since the salt ions have a much greater charge density than the proteins. So as the salt is added and these small ions bind water molecules, the protein molecules are forced to interact with themselves and begin to aggregate So when enough salt has been added, the proteins will be begin to precipitate. If this is carried out at a cold temperature like in ice, the proteins will precipitate without denaturation. Thus, the proteins can be collected by centrifugation and then redissolved in solution using a buffer with low salt content. This process is called "Salting Out" and works best with divalent anions like sulfate, especially ammonium sulfate which is highly soluble at ice temperatues

Ammonium sulfate fractionation is referred to as an ammonium sulfate “cut”: Add ammonium sulfate to a point below which the protein of interest precipitates. Centrifuge to pellet unwanted proteins. The protein of interest is in the supernatant. Add additional ammonium sulfate to the supernatant to precipitate the protein of interest. Centrifuge to obtain the protein of interest in the pellet. Proteins that are more soluble will remain in the supernatant Precipitation or “salting out” of proteins with high salt concentrations is used for the bulk fractionation of proteins. The method is simple, applicable to large volumes and usually does not effect protein activity. Ammonium sulfate is usually the precipitating salt. Ammonium Sulfate Fractionation or “Salting Out”

Dialysis Following a salting-out step, the solution will contain a high concentration of salt that can be disruptive to subsequent chromatographic steps. The salt can be removed by dialysis dialysis tubing has pores with a specific molecular weight cut-off that allows smaller molecules (salt) to pass.

Dialysis: It is a very common method which uses semi permiable membrane for equilibration of two solution across the membrane. An aqueous solution containing both macro and micromolecules is placed in a tubular cellophane bag which in turn is placed in a given buffer or simple water. Small solute molecules and water freely pass through the bag, but molecules larger than the pore size of the membrane are confined to the tubing. It causes either dilution or concentration non dialysable materials until equilibrium is reached. The external fluid must be repeatedly changed to maintain the required concentration of macromolecule inside the bag.

Membrane separation: -Microfiltration: µm, bacterial and yeast cells. -Ultrafiltration: macromolecules (2000 <MW< 500,000) -Dialysis: removal of low-MW solutes: organic acids (100<MW<500) and inorganic ions (10<MW<100). -Reverse osmosis: a pressure is applied onto a salt- containing phase, which drives water from a low to a high concentration region. MW < 300.

The rate of dialysis depend on the concentration of solution of both sides of the membrane. As the molecules move through the membrane, the concentration decreases in the higher molecular side and increases in the lower molecular side. Mixing occur by diffusion and convection so that a small amount of concentration difference is maintained. To maximize the rate of movement it is necessary to agitate the liquids.

Dialysis depend on a number of factors a)Pore size of the membrane b)Size c)Charge of the dialyzable molecule It is often very slowly and the rate can be improved considerably if the ionic strength of the solution is increased. The time required for the first ten fold decrease is much less than that for the second tenfold decrease. Changing the external fluid after each ten-fold decrease an solve the problem.