Section 4.2
Salting out: Ammonium Sulfate Precipitation
Background
Proteins vary greatly in their solubility depending on their amino acid composition and the characteristics of the solvent such as ionic strength and temperature. A common approach for separating proteins is to add increasing concentrations of a salt in order to “salt out” the proteins. Salting out is often used as an early step in a purification strategy. Ammonium sulfate is commonly chosen as the salt since high concentrations can be achieved and since it does not usually denature proteins. Even if a significant increase in purity is not achieved, an ammonium sulfate “cut” can be used to concentrate a large volume of extract for the subsequent purification steps.
“Salting out” is thought to work by “dehydrating” the environment around the protein. When ammonium sulfate is added to the protein solution, a large number of water molecules bind to the sulfate ion, which reduces the amount of water available to bind to the protein. If a protein is not hydrated by binding to water molecules, it will precipitate. Different proteins precipitate at different concentrations of ammonium sulfate. We can take advantage of this difference to remove some contaminating proteins from our crude extract.
In this ß-galactosidase purification, we will use precipitation with ammonium sulfate as our first step in the purification process. We know from published papers that ß-galactosidase precipitates when the concentration of ammonium sulfate reaches 33% of saturation. (We could also determine this on our own by trial and error.) It has been determined that 100 ml of water is saturated with ammonium sulfate when it contains 70 g. 33% saturation would be 23.1 g per 100 mls or 231 mg per ml. That is the amount we will use in step 3 below.
Procedure 4.2
Ammonium Sulfate Precipitation
- Pour the cell free extract (the supernatant from procedure 4.1) into a beaker with a stir bar, allowing for a 50% volume increase. Prepare an ice bath with water (an ethanol ice bath can actually freeze the sample) to contain the beaker with the sample in it.
- Add crystalline ammonium sulfate slowly over a period of 30-45 minutes with gentle stirring, adding a total of 231 mg (NH4)2SO4 per ml of starting solution. Keep your sample on ice at all times.
- Add 1 ml of 1 M NaOH for every 10 grams of ammonium sulfate. Let stirring continue for 1-3 hours.
- Split the volume of your sample equally into two centrifuge tubes so that you can balance them against each other. (Why?) Centrifuge the entire sample volume at 10,000 xg for 10 minutes. Use the swinging bucket rotor with 40 ml centrifuge tubes. (If you use larger bottles, the pellet can spread out too much to recover easily.)
- Although the ß-galactosidase activity should be in the precipitate, decant and save the supernatant. Refrigerate the supernatant after labeling it. ( It is always good practice to save both the supernatant and precipitate until you verify the location of your target protein)
- Resuspend the AS pellet in 1/10 the original volume of breaking buffer (step 4.2).
- Remove 0.2-0.4 ml in 0.1 ml aliquots for later assay and analysis steps.
- The resuspended pellet may be stored at 4°C. (Did you remember to record the volume of resuspended pellet?) The entire volume of the AS pellet will be used in the dialysis step in procedure 4.3 below. Therefore, you need to save aliquots before moving on to dialysis.
Assays
- Perform enzyme activity assays. Calculate the % of total units in the pellet and in the supernatant. Determine the % yield.
- Once you have determined that you have enzyme activity in your AS pellet, proceed with the next step in the purification, dialysis, Procedure 4.3. As time permits (after dialysis is started), determine the protein concentration using the Bio-Rad assay and calculate specific activity according to the assay methods in Chapter Two.
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