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Amonium Sulfate Precipitation APS of Proteins Problem

Amonium Sulfate Precipitation APS of Proteins Problem - Recombinant Protein Forum

Amonium Sulfate Precipitation APS of Proteins Problem - Recombinant Protein Forum. Discuss purification, expression, and isolation of recombinant and cloned proteins here.


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Old 07-29-2008, 03:12 AM
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Question Amonium Sulfate Precipitation APS of Proteins Problem



hello, I have made my protein in bacteria in a batch of 2 L.
There is protein activity however I would like to concentrate my enzyme with APS precipitation.

So far I cant pellet or precipitate it out! Has anyone had this problem?
what can i do?

thanks
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Old 12-23-2008, 01:54 PM
tgd tgd is offline
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Default Re: Amonium Sulfate Precipitation APS of Proteins Problem

Hello,

I think we need more details. What is the percentage saturation with ammonium sulphate? How are you trying to collect the percipitate? Are you centrifuging? For how long, and what is the g-force?

Normally, I centrifuge at 16 000 g for 30 min.

For example, bring your enzyme preparation to 30% saturation by adding solid ammonium sulphate slowly over a period of 30 min. (You may wish to consult an ammonium sulphate chart to do this. An example is given in Dawson, 'Data for Biochemical Research'. You will need to add about 176 g/L. If you are not consulting a chart be very careful of volume changes).

*Then continue to stir for at least another 30 min*. Centrifuge as above and collect the pellet. Resuspend in a minimum volume of buffer (you may wish to use a Potter-Elvejhem homogenizer) and check if your enzyme is present.

Let's say there is none there.

Bring the supernatant from the centrifugation step (that is the 30 percent supernatant) to, say, 65 percent ammonium sulphate as outlined above and spin again. Resuspend the pellet in a minimum volume of buffer and check for the presence of enzyme. Lets say it has precipated

This is a (30-65)% cut. You may need to modify this, or do, say, a (0-65)% cut if it is just enzyme concentration you are after.

Finally, everthing should be done at 4 degrees C.

As far as I am concerned, this this the very best reference:

Dixon, M. & Webb, E. C. (1961). Enzyme fractionation by salting-out: a theoretical note. Adv. Protein Chem. 16, 197 - 219.

Good luck,

tgd
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