December 07, 2022, 02:57:35 AM
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Topic: Is it possible to get a 100% pure enzyme after fractionation and dialysis?  (Read 4132 times)

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Offline Kalibasa

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Of course I would guess no, but I'm struggling to explain why- we need to "include techniques that either were already performed or should be done to support your claim." We did this in the lab:

Salt fractionation with 35% ammonium sulfate
Salt fractionation with 57% ammonium sulfate
Methanol fractionation and heat treatment
Dialysis

I don't see how any of these can give hints as to the final purity of the enzyme, and I can't seem to find much in outside sources. If someone could even give me a hint (like what terms I should use in a search engine or a book index), it would help A LOT :)

My only guess is that, in the context of our lab, we still haven't done absolutely everything we can to purify the enzyme. In sources I saw a lot of people mentioning ion separation and gel electrophoresis as other ways to purify an enzyme. Could I say that our enzyme is not 100% pure because these techniques haven't been performed? Or is there something I'm just missing?

Offline Yggdrasil

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Well, think about the techniques you use.  On what basis do they purify your enzyme?  Of the possible contaminants, which ones will your techniques remove and which ones will they not have removed?

Offline Kalibasa

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It should have removed the cell debris in the initial centrifugation, many of the other proteins in the heat treatment and the fractionations and the ions and small molecules in the dialysis. What is left- sugars and larger polar molecules?

My lab was due today and I had no idea what to write- I went with "We never performed ion-exchange chromatography or gel filtration." Would you say that was right?

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