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I. Introduction
Fractional precipitation by salt is almost invariably used at an early stage of a protein purification procedure to obtain an initial purification and concentration. Further purification is then achieved by chromatographic methods, which have higher resolving power, but generally lower capacity.
When high concentrations of salt are present, proteins tend to aggregate and precipitate out of solution. This technique is referred to as “salting out”. Protein precipitates are aggregates of protein molecules large enough to be visible and to be collected by centrifugation. The distribution of hydrophilic and hydrophobic residues at the surface of a protein determines its solubility properties. When proteins are in solution, water molecules form a shell over hydrophobic patches on the protein. The water is ordered in the shell so as to maximize hydrogen bonds. This ordering is energetically unfavorable. At high salt concentrations, water is pulled from the hydrophobic patches to associate with salt ions. This forces hydrophobic patches on the surface of the protein to interact with each other, resulting in protein aggregation or precipitation.
Ammonium Sulfate Precipitation
Salting out by adding sufficient amounts of ammonium sulfate ((NH4)2SO4) is a convenient, nondenaturing way to concentrate a protein. Protein precipitation using (NH4)2SO4 is achieved by dehydration in the microenvironment of the protein molecule. In solution, a large number of water molecules are bound to the sulfate ion (SO42-), reducing the amount of water available to interact with the protein molecules. At a particular concentration of (NH4)2SO4, an insufficient quantity of unbound water will remain to keep a given protein species in solution, resulting in the precipitation of that protein. A saturated (NH4)2SO4 solution in water is ~ 4M with a density of 1.235 g/ml compared to a protein aggregate density in this solution of 1.29 g/ml. This difference makes it possible to pellet out the aggregates by centrifugation.
II.Materials and Methods
Materials:
1. Egg white 蛋白溶液
2. Saturated ammonium sulfate solution (75 g in 100 ml of water)
3. 1% NaCl
Procedure:
Preparation of crude egg albumin.
1. Add 0.5 ml saturated ammonium sulfate solution to 0.5 ml egg white drop by drop, votex to mix between addition. If the salt is added too quickly, then high local concentrations will develop and proteins will be precipitated that would remain soluble at the target ammonium sulfate concentration. Such proteins may not readily re-dissolve, and reduce the effectiveness of fractionation.
將0.5 ml的飽和硫酸銨溶液，逐滴加入0.5 ml的蛋白溶液中，每加入一滴硫酸銨溶液後，為避免局部硫酸銨濃度過高，請立刻混和均勻。[請注意操作蛋白質溶液時，應避免出現起泡的現象，泡泡的表面張力會將蛋白變性。]
2. The mixture is allowed to stand on ice for 30 min to allow protein precipitation taking place.
加完硫酸銨溶液後，靜置於冰上30分鐘，讓蛋白沉澱。
3. Centrifuge at 12000 rpm for 5 min. Remove the supernatant by pipette as much as possible.
以12000 rpm的離心力離心5分鐘，將上清液以微量吸管盡可能的吸乾，但是不要碰觸到蛋白沉澱。
4. Add 1 ml of 1% NaCl to resuspend the pellet (crude albumin), by votex or pipetting. Remove any undissolved material by centrifuge. (12000 rpm, 5 min)
加入1 ml 1%NaCl，以微量吸管輕輕的反覆沖洗蛋白沉澱，使其重新溶解；如無法完全溶解，最後再利用12000 rpm的離心力離心5分鐘，將少許的不溶物移除。
III. Results:
1. Prepare 1 ml of 5% and 2.5% crude albumin each for Exp. 2.2
請準備1 ml 5%和2.5%粗白蛋白溶液，實驗2.2將會用到。
2. Transfer 400 ul, 400 μl, and 50 μl of crude albumin to three separate eppendorf tubes. Label the tubes with group number and store at -20℃ freezer for the chromatography separation (Exp. 2.2 and 2.3) and gel electrophoresis later.
將400 μl, 400 μl, 與50 μl 的粗白蛋白溶液，分裝到3個1.5 ml微量離心管，請在管外標明你的組別號碼，保存到零下20度的冰箱，在接下來的實驗2.2、2.3、3將會用到。
2.1: Protein Fractionation is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by LibreTexts.
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