About the ammonium sulphate precipitation? - prootein purification (May/05/2007 )

Since the potassium phosphate and sodium chloride are readily available and cheaper chemcials than ammonium sulphate. Can er make use of these salts to substitute ammonium sulphate for the proein precipitation? and tell the reason, thanks

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Yes, you can, just those salts won't be as much effective and safe for your proteins as ammonium sulphate.

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you may precipitate proteins with any substance that stronger bind water than proteins; I suppose that ammonium sulphate ionically/molecularly interacts with proteins and prevents denaturing even during the precipitated state of proteins;

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You are right, sulphate anions stabilize protein structure. That's exactly what I ment by "safe for protein".

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why only sulphate anions can stabilize the protein but the others such as phosphate and chloride?

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Anions and kations are ordered in so-called "Hofmeister series" - from most to least effective in salting-out and stabilizing of protein structure. Eg.:
anions --- citrate > sulfate > phosphate > chloride > nitrate > thiocyanate
cations --- ammonium > potassium > sodium > lithium > magnesium > calcium
(Order of ions may be different in other sources.)

I don't know how it exactly works and don't have enough time and sources to search in but there are few words on WikiPedia so do search by yourself.

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excellent explanination,
thanks
i am fully understanding

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Here is the theory of Ammonium sulfate precipitation from Wikipedia

"Ammonium sulfate precipitation is a method of protein purification by altering solubility of protein. It is a specific case of a more general technique known as salting out.

Ammonium sulfate is commonly used as its solubility is so high that salt solutions with high ionic strength are allowed.

The solubility of proteins varies according to the ionic strength of the solution, and hence according to the salt concentration. Two distinct effects are observed: at low salt concentrations, the solubility of the protein increases with increasing salt concentration (i.e. increasing ionic strength), an effect termed salting in. As the salt concentration (ionic strength) is increased further, the solubility of the protein begins to decrease. At sufficiently high ionic strength, the protein will be almost completely precipitated from the solution (salting out).

Since proteins differ markedly in their solubilities at high ionic strength, salting-out is a very useful procedure to assist in the purification of a given protein. The commonly used salt is ammonium sulfate, as it is very water soluble and has no adverse effects upon enzyme activity. It is generally used as a saturated aqueous solution which is diluted to the required concentration, expressed as a percentage concentration of the saturated solution (a 100% solution).

In the preliminary test, the ammonium sulfate concentration is increased stepwise, and the precipitated protein is recovered at each stage. Each protein precipitate is dissolved individually in fresh buffer and assayed for total protein content and amount of desired protein. The aim is to find the ammonium sulfate concentration which will precipitate the maximum proportion of undesired protein, whilst leaving most of the desired protein still in solution.

The precipitated protein is then removed by centrifugation and then the ammonium sulfate concentration is increased to a value that will precipitate most of the protein of interest whilst leaving the maximum amount of protein contaminants still in solution. The precipitated protein of interest is recovered by centrifugation and dissolved in fresh buffer for the next stage of purification.

This technique is useful to quickly remove large amounts of contaminant proteins, as a first step in many purification schemes. It is also often employed during the later stages of purification to concentrate protein from dilute solution following procedures such as gel filtration."

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Hihi all...i'm a newbie in this procedure. The posts above was quite informative Just a question though,
what would be the effect of usng a narrow cut/concentration of ammoium sulphate on protein precipitation? Eg 30-50% vs say 40-50%? I think the yield would decrease but would the purity of the protein increase?

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this is exactly the point: You have to find the line of compromise between a good yield (recovery) on the one hand, and losing bulk proteins on the other hand; you have to do some pre-experiments to find the right conc of ammonium sulphate to find your best condition (depends on your protein annd the type of cell/tissue)

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