Dear all,
I am trying to get rid of imidazole in a purified protein sample (usually 3ml sample), and concentrate it as well. So, I am thinking of using ammonium sulphate method for such a small sample. Please suggest me a protocol.
many thanks
You can usually remove the imidazole by dialyzing the protein solution against a buffer or passing it through a gel filtration column or desalting column. Ammonium sulfate precipitation should not be necessary, but here is how you do it.
Most proteins precipitate with ammonium sulfate if the concentration is high enough. For example, some enzymes can be purchased as suspensions in a 3.2 M ammonium sulfate solution. The solid ammonium sulfate is added to the cold protein solution to achieve the final desired concentration (accounting for the extra volume contributed by the ammonium sulfate, see the table) and gently mixed in until dissolved. After storage for a while at 4oC, the protein precipitates. It can be stored in the refrigerator in this state for a long time. It can then be recovered by centrifugation. The pellet is resuspended in the desired buffer. At this point, the protein solution has a lot of ammonium sulfate in it, so it is usually desirable to dialyze the protein or pass it through a gel filtration column.
https://www.denovobiotech.com/custom-proteins/technical-information-proteins/ammonim-sulfate-fractionation-for-protein-purification
DEar Mark Ashraf Fahmy
my prefered approach for a such range of volumes is desalting with PD-10 coloumn (which slighty dilute the sample) followed by concentration by ultrafiltration if required.
if you are interested in the following link you can see a video about the use of PD-10
https://www.blogger.com/video.g?token=AD6v5dwNlVHz3BclDT9RLY-6KbEEqkS3RnRdHn4BqTsZW4j_prf3KZwHDMTzRYT-B9Yifvmde7RdExL5AArLFtg1Q13fu4-83o3M8bCka1jJ5PfWFK8B53BOpx6YrzvLr2qDiPgT15c
ammoinun sulfate precipitation may also be a way to remove imidazole but you will replace it with traces of ammoniun sulfate that will remain in your sample, and it is not a real buffer exchange approach.
good luck
Manuele
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