Lab Activity 11 Purification of LDH Part II IUG, Fall 2012 Dr. Tarek Zaida

Background Depending on the protein of interest to be purified, there are various methods that can take advantage of these properties of protein. The table below lists some of the general properties of proteins that can be useful for protein purification.

Ammonium Sulfate Precipitation In many cases, cell lysates can be loaded directly onto chromatography columns. However, in some cases other molecules present in the lysate interfere with binding of the protein to the resin. In addition, some resins (especially affinity resins and sepharose-based resins) are fairly expensive; loading crude cell lysates on these columns may result in binding of cellular material (e.g. lipids and DNA) that are difficult to remove, and which may damage the column. As a result, purification methods often begin with one of several possible simple techniques that remove at least some of these unwanted materials prior to using an expensive column.

One of the most commonly used crude purification techniques involves the use of differential solubility. Proteins precipitate with increasing ammonium sulfate concentrations, with most proteins precipitating somewhere between 10% and 60% ammonium sulfate. (The percentages are relative to a saturated solution, which has a concentration of about 4 M; Thus most proteins precipitate between 0.4 M and2.4M) This can allow a simple, partial, purification of a protein; If the protein of interest precipitates at 40% ammonium sulfate, many other proteins will remain in solution, as will many other non-protein molecules.

Most proteins are not damaged by ammonium sulfate precipitation, and can be resuspended in a small volume of buffer. Ammonium sulfate precipitation results in a high salt concentration in the protein solution; this may be advantageous (if the intended next step is hydrophobic interaction chromatography), or deleterious (if the next step is ion exchange chromatography). When necessary, two methods are frequently used to remove the salt. 1. One method is gel filtration chromatography. 2. Another frequently used method is dialysis.

Dialysis Dialysis involves placing the protein solution in a semi-permeable membrane, and placing the membrane in a large container of buffer. Small molecules (such as salt ions) pass through the dialysis membrane (moving from high concentration to low concent-ration), while large molecules are unable to cross the membrane. In principle, dialysis could allow separation of large proteins from small ones; In practice, however, the pores in the tubing are insufficiently uniform to allow this technique to be used effectively.

Illustration of A dialysis membrane

Chromatographic methods Most purification methods involve chromatography. Chromatographic methods involve a column of an insoluble material that can bind molecules based on specific properties common to proteins. The solution containing the mixture of proteins is then allowed to pass through the column; the protein of interest may bind (depending on its properties), while at least some impurities remain in solution and leave the column. The procedure is completed by eluting (i.e.“removing”) the proteins that have bound to the column.

Ammonium Sulfate Precipitation 1. Slowly(over a period of ~15 minutes) add 0.39 g of ammonium sulfate per ml of supernatant to your filtered supernatant. It is best to perform this step in the cold room on a magnetic stirrer (obviously, you need to put a stir bar into your sample). Avoid stirring too violently (proteins denature if subjected to shearing stresses; if you see bubbles forming, you are denaturing your proteins). Stir for an additional 15 minutes after you finish adding the ammonium sulfate (this gives the ammonium sulfate a chance to dissolve, and allows the proteins a chance to equilibrate to the presence of the ammonium sulfate).

2. Centrifuge the sample (as before) 2. Centrifuge the sample (as before). Pour the supernatant into a separate container while keeping the pellet in the centrifuge tube. Save both supernatant and pellet in the refrigerator until the next lab period. The LDH should be in the pellet.