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Which of the following methods would yield the most purified protein fraction?

A. Salt precipitation

B. Charge separation

C. Affinity purification

The first one is the most commonly used, is it the best ?

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The choice of strategy for protein purification of course varies by application. As @A.Kennart points out, affinity chromatography is the most popular right now. It can be expensive. If you have an expression system for a recombinant protein its more common than not to add a poly-Histdine tag on the N or C terminus or some other protein fusion. So there are cases where you won't be able to use it.

For instance if the protein you are looking for is not coming from a recombinant source / is not sequenced, it can get expensive and time consuming - getting an antibody made requires that you have a somewhat pure prep of the protein in the first place.

The most common reason we still use all these methods are because different levels of purity and different raw materials show up in different contexts.

In the case where the protein has never been isolated before or you have a sample (like a cell sample rich in a particular protein) then salt and sizing columns are very useful. Salt precipitation is useful when you have a lot of sample and you need to make some rough cuts. Even if the gene is well characterized, it may depend on expression in its native organ.

I've prepped protein using ammonium sulfate precipitation from homogenized whole tissue for instance. That's great - the result got rid of well over 90% of the material, but the outcome has hundreds of bands on the gel. In this case an affinity column was used next; immobilized conconavalin A would bind to the glycosides on the target product. If I had put the whole tissue homogenate I would have destroyed the affinity column by stuffing it full of meaty gunk.

Charge affinity columns with salt gradients are often still needed after affinity chromatography. Some folks don't need pure protein, but affinity chromatography (esp his tags) will still have many bands left on a protein gel and those contaminants always leave your experiment in question.

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    $\begingroup$ thanks :) my first tech job involved collecting adrenal glands at a slaughterhouse. they did not smell or look good - no way you'd put that on a $300 column! Salt and centrifuge. was the only way to clean that up. $\endgroup$ – shigeta Nov 15 '13 at 3:03
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    $\begingroup$ Well, I see that this is the accepted answer, and I don't disagree with any of it, but it isn't really the answer to the question that was posed. In terms of a purification factor (e.g. increase in specific activity of an enzyme), affinity purification will win out every time. $\endgroup$ – Alan Boyd Nov 15 '13 at 16:06
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In my protein purification experience, affinity purification is the most selective purification strategy. This makes sense because affinity tends to be very specific, and only one protein should in theory be preferentially separated by the column. Think how specific the antibody-antigen interaction is compared to the differences in, say, charge between proteins (which could be very slight). When I was purifying proteins I would pretty much always start with affinity purification. If more purity was desired after the affinity purification step, I could perform salt precipitation or charge separation on the affinity-purified sample, which had already been cleared of major contaminants/nuisance proteins by affinity purification.

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    $\begingroup$ The fact that all of the tagging methods used for purification of proteins expressed from cloned genes (his tags, FLAG tags etc.) are designed to facilitate affinity purification approaches underscores this answer. $\endgroup$ – Alan Boyd Nov 14 '13 at 19:30
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If you stick with only one technique you get high yield but low purity. With each additional step you lose some sample, but gain on purity. The whole process is a trade off and the answer depends on your purpose of purification.

Affinity chromatography is somewhat different from other techniques you listed. Most people tend to choose it as the most effective one. It is based on specific high affinity binding. The problem arises when you start eluting the bound protein. The usual practice is using low pH and compensating it with high pH just after elution. There is a high probability that drastic pH changes are going to disrupt protein activity/native conformation. Another possibility is immobilized metal affinity chromatography if your protein is expressed recombinantly, but that is not the case usually and it was explained in detail in previous answers.

My approach would be to start with salt precipitation to remove bulk proteins. Proceed by ion exchange chromatography (depending on the properties of the target protein, optimal if its pI differs greatly from other proteins in the solution, but possible otherwise with some difficulties). The final purification step should be size-exclusion chromatography for further purification. This is only a template of the most commonly used purification approach. Therefore, the exact purification procedure depends on your target protein, but I recommend this strategy as a starting point.


To answer your question directly, affinity chromatography would yield the most purified protein fraction, but that would be a simple answer to a really complex question.

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  • $\begingroup$ If possible please add some references to that users can look them up if needed. $\endgroup$ – WYSIWYG Dec 21 '16 at 8:08

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