Dear researchers,
May I know what is the advantage of using ammonium sulphate to do protein extraction?
Thank you.
Ammonium sulfate is often used as an initial purification step. You can precipitate proteins from an extract with high concentrations of ammonium sulfate without causing denaturation. By gradually adding the ammonium sulfate a little at a time, you can achieve some purification of a protein by differential precipitation.
- protein purification and fractionation that can be used to separate proteins by altering their solubility in the presence of a high salt concentration.
In addition to what was said by Adam B Shapiro , you can use ammonium sulfate for the purification/enrichment of untagged (no His, GST, MBP, etc.) proteins. This is really important if you want to work with a protein without altering its amino acid sequence. It is also super reproducible: the same protein in the same buffer and temperature will always precipitate at the same ammonium sulfate concentration. And its cheap :)
To add some practical meat onto the theoretical skeleton others have provided:
In an initial experiment, you add ammonium sulphate to small aliquots (say, 500 µL) of your lysate to achieve 5%, 10%... saturation. Incubate for 30 min on ice, gently shaking occasionally. Then spin down any pellet and separate from the supernatant (be careful to balance the rotor!). Determine how much of your protein of interest is in the pellets and supernatants (if you have an antibody use a spot blot for quick results). You want to identify two concentrations, where the PoI is just soluble (all in the supernatant, only background in the pellet) and where it is insoluble (all in the pellet).
For the actual purification, you slowly (!!) add AS to the former concentration (there are tables for that, for example http://cshprotocols.cshlp.org/content/2006/1/pdb.tab2), stir for 30 min and then spin down the irrelevant proteins (but keep all fractions until purification is complete!).
Then bring the supernatant to the latter concentration, incubate and spin. The pellet will contain most of your PoI.
Several points are important:
- Keep everything on ice. AS is the more denaturing the higher the temperature. Cold rooms are effective for chilling operators, but not samples.
- AS works by lowering the available water concentration, this reduces movement within a protein. Thus, AS increases crystallisability and stability, but lowers enzymatic activity.
- Residues of AS cause corrosion of rotors, especially those of aluminium. Carefully clean and dry them before putting them back into the cupboard.
- Carefully determine volume, protein concentration and concentration of your PoI in all fractions. This allows you to calculate recovery and enrichment of all your purification steps.
- AS as salt interferes with electrophoresis. Chloroform/methanol precipitation (doi:10.1016/0003-2697(84)90782-6) removes such interference.
- AS is almost non-toxic (as nothing is absolutely non-toxic), but large amounts are required (in one lab I used to work in we bought it in 50 kg boxes). Check how much you can put down the drain without making your local sewage treatment plant keel over.
having an excellent benefit in biological research for making of solution mixture, maintaining pH, referred following article
Influence of ammonium sulphate feeding time on fed-batch Arthrospira (Spirulina) platensis cultivation and biomass composition with and without pH control
Article Influence of ammonium sulphate feeding time on fed-batch Art...
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