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Purifying a "protein"

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#1 Runnergirl22



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Posted 30 December 2012 - 05:42 PM

Hi all,
I'm a nurse taking a biochemistry class. I have limited knowledge on the subject and was presented with the following assignment:

Enzyme X is a highly pigmented protein that imparts the characteristic color to certain blue-green algae. It also facilitates a reaction necessary to the survival of this species; we can follow the kinetics of this reaction by measuring the conversion of Substance X to Substance Y at various times during purification.
1. Given a pure preparation of these algae, and the required supplies and equipment, devise and outline an empirical procedure for purifying Enzyme X.
2. What is a good indication of purity in your preparation?

Correct me if I'm wrong, but wouldn't it be hard to purify "enzyme x" given the limited amount of information the above scenario presented?

I was thinking of responding with the following steps...could you please critique?

1. Centrifigation
2. Dialysis
3. gel filtration
4. Test for levels of enzyme x by testing to see which sample had the greatest conversion levels to substance y
5. gel electrophoresis
6. western blot

Am I on the right track, or is this completely off base?

Thank you!!

#2 mdfenko


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Posted 31 December 2012 - 08:05 PM

it would not be hard with the info given. it is easier to follow the purification of a colored protein and you have a specific assay for it.

as for your scheme:

centrifugation will pellet the algae but you also have to lyse the cells. dialysis is mostly for buffer exchange (following other procedures).

gel filtration early in the procedure will eliminate bulk amounts of proteins outside the range of the protein of interest's molecular weight. it is often used for final polishing during a purification (it is easy to follow with a colored protein).

you might want to consider: ammonium sulfate and/or acetone fractionation, ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography (group specific and/or substrate), immunoaffinity chromatography (if an antibody is available).

gel electrophoresis and western blot are generally used to determine the presence and purity of a protein, not as purification steps (page can be preparative).

(i have purified colored proteins from blue-greens, but it has been more than 35 years since)

Edited by mdfenko, 31 December 2012 - 08:08 PM.

talent does what it can
genius does what it must
i used to do what i got paid to do

#3 Pangea



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Posted 13 January 2013 - 07:12 AM

Run a SDS-PAGE and do Silver-Staining. Measurement on 280 nm for Purity.

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