Blog: Protein Aggregates
Nov 2011 | Dave
A protein purification classic: Ammonium Sulfate Precipitation
An underutilized way to purify proteins
In the 'old days' - i.e. when protein purification meant going to the slaughterhouse to get raw materials such as placenta, brain or blood - the first real purification step was an ammonium sulfate precipitation. Of course, the natural tissue needs to be homogenized and solids removed by filtration or centrifugation. This first clarified feedstock contains a multitude of protein solutions, not that different from a modern-day E.coli lysate. Such lysates can be fractionated according to the proteins' tendency to precipitate with increasing ionic strength. Turns out that Ammonium Sulfate is a particularly suitable precipitation reagent for this purpose. In most cases, adding a portion of a saturated Ammonium Sulfate solution or solid Ammonium Sulfate Salt turns the solution cloudy, indicating the precipitation of certain protein fractions. In most cases protein purification researchers try to avoid their protein solutions turning cloudy. In this case however, precipitation is the goal. In fact, after spinning down the precipitate, the the Ammonium Sulfate concentration can be further increased, resulting in additional precipitation that can be removed by centrifugation. This procedure can be repeated several times, yielding a number of Ammonium Sulfate fractions. At this point samples are taken (precipitate and clarified solutions from every precipitation step) and are analyzed. Since the tendency of different proteins in the lysate differs as a function of Ammonium Sulfate concentration, individual fractions typically contain fewer and fewer proteins.
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How do you use this data to establish a purification procedure for a particular target protein? In the best case scenario you select the fraction that precipitated the protein of interest. Then you'd try to bring this precipitate back into solution, for instance by reducing the Ammonium Sulfate concentration and adjusting the buffer composition. Dialysis is a simple way to achieve this. Once clarified, further purification steps can be added if necessary. In cases where the precipitated protein does not re-solubilize, the 'earlier' fraction should be chosen, i.e. the lower Ammonium Sulfate concentration fraction that contains the protein in a non-precipitated form. This fraction contains more contaminants, but has fewer contaminating proteins than the starting material. |
Practical tips for Ammonium Sulfate precipitation experiments:
• Carry out the precipitation reaction on ice (or thermostatted at a specific temperature), in a glass beaker with a magnet bar, on a stir plate
• Sequential operation: for each fractionation step let the precipitation reaction come to completion by incubating for a minimum of 10 min. This will procedure may yield incomplete precipitation, but is much faster
• Parallel operation: prepare multiple precipitation experiments, adding a final Ammonium Sulfate concentration of 10%, 20%, , 30% etc. Over night incubation assures that the precipitation has come to completion.
• Working with solid Ammonium Sulfate allows to explore high Ammonium Sulfate concentrations:
- Dry Ammonium Sulfate by baking overnight at 120C and grinding to a fine powder (wear a dusk mask!)
- Add the Ammonium Sulfate powder very slowly. Don't add entire chunks, avoid clumping in the stirred solution. Sprinkle in the powder and wait for it to dissolve completely.
- Add pre-weighted amounts of ammonium sulfate.
• Working with Ammonium Sulfate is more convenient. Just prepare a saturated Ammonium Sulfate solution by adding 750 g of Ammonium Sulfate to 1 liter of destilled water and stir for 15 minutes. The supernatant of this solution is 100% saturated.
• Local Ammonium Sulfate concentration spikes can be avoided by working with a solution. However, a saturated solution dilutes the lysate, increases working volume and limits the Ammonium Sulfate concentration to the low Ammonium Sulfate concentration regime. Regardless, add the Ammonium Sulfate solution slowly and stir vigorously.
• Avoid foam formation or frothing (this can oxidize the protein)
• Take samples of the solution and the precipitate for analysis of protein composition
• Dilute the precipitate with buffer to the original lysate volume to allow for better comparison of yields between fractionation steps
Useful online tools for Ammonium Sulfate precipitations:
Online calculator to calculate the amount of solid Ammonium Sulfate required to add to a specific volume of a solution to get a specific percentage saturation at a specific temperature;
Ammonium Sulfate precipitation demonstration experiment for the isolation of Hemoglobin, Amylase and Protease by ammonium sulfate precipitation
Ammonium Sulfate precipitation at work: recent examples of the utility of this economic protein purification method.
Mahn A, Ismail M. Depletion of highly abundant proteins in blood plasma by
ammonium sulfate precipitation for 2D-PAGE analysis. J Chromatogr B Analyt
Technol Biomed Life Sci. 2011 Nov 15;879(30):3645-8.
Moore PA, Kery V. High-throughput protein concentration and buffer exchange:
comparison of ultrafiltration and ammonium sulfate precipitation. Methods Mol
Biol. 2009;498:309-14.
Park JW, Lee SG, Song JY, Joo JS, Chung MJ, Kim SC, Youn HS, Kang HL, Baik SC,
Lee WK, Cho MJ, Rhee KH. Proteomic analysis of Helicobacter pylori cellular
proteins fractionated by ammonium sulfate precipitation. Electrophoresis. 2008
Jul;29(13):2891-903.
Ko KY, Ahn DU. Preparation of immunoglobulin Y from egg yolk using ammonium
sulfate precipitation and ion exchange chromatography. Poult Sci. 2007
Feb;86(2):400-7.
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Protein Aggregation: a nuisance
I've experienced it many times in my own research project, typically at the end of a week-long effort to express and purify a particular target protein. If not at that stage then as an outcome of crystallization experiments that I had set up. Most crystallization drops contain either precipitate or they remain clear. The 'clears' have always fascinated me (besides the drops with crystals of course) because there was direct evidence that I can tweak the protein environments in ways that keep the protein soluble for weeks and months at room temperature. So I knew, there is a way. I suspect that there's a way to keep practically any protein in solution and accessible to assays, storage and biophysical inspection.
I suspect that for every protein with a specific sequence there is a 'sweet spot'. The way to find this sweet spot is systematic variation of the solvent properties and testing the effect on the protein. This is what this product / blog is about.
I'd like to provide a forum/product toolset that lets you find this sweet spot.
Take care,
Dave
